Sebastian Fiedler

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins.

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).

Protein folding in membranes

Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of α-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today’s life sciences. Here, we review recent progress in the folding, unfolding and refolding of α-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.

Mia40-dependent oxidation of cysteines in domain I of Ccs1 controls its distribution between mitochondria and the cytosol

Sod1 is an important antioxidant enzyme that becomes activated by its chaperone, Ccs1. The localization of Ccs1 to mitochondria is controlled by the oxidoreductase Mia40. The formation of a disulfide bond between Cys-27 and Cys-64 in Ccs1 is critical for import and stability but not for Ccs1 activity in the maturation of Sod1.

Amyloid Precursor Protein Dimerization and Synaptogenic Function Depend on Copper Binding to the Growth Factor-Like Domain

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

Automated Circular Dichroism Spectroscopy for Medium- Throughput Analysis of Protein Conformation

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For peptides and proteins, it is highly sensitive to changes in secondary structure, which may be caused by alterations in amino acid composition or solution conditions (e.g., temperature, pH, salts, detergents, denaturants, and excipients), post-translational modifications, self-association, or ligand binding. The assets of CD spectroscopy are that the signal is directly linked to structure, the analyte is measured without labels and in solution, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer equipped with a low-birefringence flow-through cell that is coupled to a three-axis robotic liquid-handling system. This enables unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We show that the accuracy, precision, and reproducibility afforded by the new instrument are excellent and exemplify how the advantages offered by automated CD spectroscopy can be exploited to quantify protein stability by titration with chemical denaturants.

Real-Time Monitoring of Membrane-Protein Reconstitution by Isothermal Titration Calorimetry

Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K+ channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-β-d-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.

The mechanism of denaturation and the unfolded state of the α-helical membrane-associated protein Mistic.

In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, α-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the α-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an α-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

Preparation of ready-to-use small unilamellar phospholipid vesicles by ultrasonication with a beaker resonator

Lipid vesicles are widely used as models to investigate the interactions of proteins, peptides, and small molecules with lipid bilayers. We present a sonication procedure for the preparation of well-defined and ready-to-use small unilamellar vesicles composed of phospholipids with the aid of a beaker resonator. This indirect but efficient sonication method does not require subsequent centrifugation or other purification steps, which distinguishes it from established sonication procedures. Vesicles produced by this method reveal a unimodal size distribution and are unilamellar, as demonstrated by dynamic light scattering and (31)P nuclear magnetic resonance spectroscopy, respectively.

Automated Circular Dichroism Spectroscopy for Medium-Throughput Quantification of Protein Conformation

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For proteins, it is highly sensitive to changes in secondary structure, which, in turn, are influenced by amino acid composition, posttranslational modifications, solution conditions (e.g., temperature, pH, salts, detergents, denaturants, excipients, etc.), and ligand binding. The CD signal is directly linked to protein structure, the analyte is in solution and label-free, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer that is equipped with a flow-through cell and coupled to a 3-axis robotic liquid handler. This enables completely unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We demonstrate that the accuracy, precision, and data quality of the automated CD spectrometer are as good as those of a conventional, manually operated instrument and exemplify how the advantages offered by automated CD spectroscopy can be exploited in protein-unfolding experiments using chemical denaturants.