Christian Hilty

Molecular Mechanisms of Ion-Specific Effects on Proteins

The specific binding sites of Hofmeister ions with an uncharged 600-residue elastin-like polypeptide, (VPGVG)(120), were elucidated using a combination of NMR and thermodynamic measurements along with molecular dynamics simulations. It was found that the large soft anions such as SCN(-) and I(-) interact with the polypeptide backbone via a hybrid binding site that consists of the amide nitrogen and the adjacent α-carbon. The hydrocarbon groups at these sites bear a slight positive charge, which enhances anion binding without disrupting specific hydrogen bonds to water molecules. The hydrophobic side chains do not contribute significantly to anion binding or the corresponding salting-in behavior of the biopolymer. Cl(-) binds far more weakly to the amide nitrogen/α-carbon binding site, while SO(4)(2-) is repelled from both the backbone and hydrophobic side chains of the polypeptide. The Na(+) counterions are also repelled from the polypeptide. The identification of these molecular-level binding sites provides new insights into the mechanism of peptide-anion interactions.

Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (transverse relaxation‐optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY‐based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence‐specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA β‐barrel from those of OmpX. Overall, the present data demonstrate that relaxation‐optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.

Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents.

For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer‐membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water‐soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein–detergent interactions in the mixed micelles. The application of paramagnetic probes to the well‐characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein–detergent micelles.

Lipid–protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and dihexanoylphosphatidylcholine (DHPC) provided a detailed description of protein–detergent interactions. The NOEs were measured in 3D 15N- and 13C-resolved [1H,1H]-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly 2H,13C,15N-labeled OmpX in micelles of DHPC at natural isotope abundance. In these mixed micelles the NMR structure of OmpX consists of an eight-stranded antiparallel β-barrel. The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 Å in height, which is centered about the middle of the long axis through the β-barrel. In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket. The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the β-barrel in a double-layer lipid membrane.

Facing and Overcoming Sensitivity Challenges in Biomolecular NMR Spectroscopy

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed.

Reversal of the hofmeister series: specific ion effects on peptides.

Ion-specific effects on salting-in and salting-out of proteins, protein denaturation, as well as enzymatic activity are typically rationalized in terms of the Hofmeister series. Here, we demonstrate by means of NMR spectroscopy and molecular dynamics simulations that the traditional explanation of the Hofmeister ordering of ions in terms of their bulk hydration properties is inadequate. Using triglycine as a model system, we show that the Hofmeister series for anions changes from a direct to a reversed series upon uncapping the N-terminus. Weakly hydrated anions, such as iodide and thiocyanate, interact with the peptide bond, while strongly hydrated anions like sulfate are repelled from it. In contrast, reversed order in interactions of anions is observed at the positively charged, uncapped N-terminus, and by analogy, this should also be the case at side chains of positively charged amino acids. These results demonstrate that the specific chemical and physical properties of peptides and proteins play a fundamental role in ion-specific effects. The present study thus provides a molecular rationalization of Hofmeister ordering for the anions. It also provides a route for tuning these interactions by titration or mutation of basic amino acid residues on the protein surface.

Bilayer in small bicelles revealed by lipid−protein interactions using NMR spectroscopy.

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.

Methanol strengthens hydrogen bonds and weakens hydrophobic interactions in proteins--a combined molecular dynamics and NMR study.

A combined simulation and experimental study was performed to investigate how methanol affects the structure of a model peptide BBA5. BBA5 forms a stable β-hairpin-α-helix structure in aqueous solutions. Molecular dynamics simulations were performed in water and methanol/water solutions using all-atom explicit models. NMR experiments were used to test the calculated results. The combined theoretical and experimental studies suggest that methanol strengthens the interactions between the polar backbone of the peptide and thus enhances the secondary structure formation; at the same time methanol weakens the hydrophobic interactions and results in an expansion of the hydrophobic core and an increase in gyration.

Nuclear Magnetic Resonance of Hyperpolarized Fluorine for Characterization of Protein-Ligand Interactions

Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the (19)F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.

Controlling membrane permeability with bacterial porins: application to encapsulated enzymes.

Recent achievements of membrane protein science allow easy protein modification by genetic engineering and, for some proteins, their production in large quantities. We regard these features as the basic requirements for applications of membrane proteins in materials science. Here, we demonstrate a possible application of membrane proteins, inserting porins from the outer cell wall of Escherichia coli into the walls of liposomes. Encapsulation of enzymes into liposomes or polymer nanocapsules protects them against proteases and denaturation. Functional reconstitution of porins into the capsule shell allows to control the rate and selectivity of substrate permeation, and thus to control the enzyme reaction kinetics. We suggest that this technique can prove to be useful in the area of biosensors, providing enzymatic stability while keeping the functionality or even enhancing the sensitivity by substrate preselection. Another application of this kind of stabilisation is in the field of single enzyme activity recording.