Inhibition of the Fibrillation of Amyloid Aβ by Hybrid-Lipid-Polymer Vesicles.

The transformation of functional proteins into amyloidic plaques is responsible for the impairment of neurological functions in patients fallen victim to debilitating neurological conditions like Alzheimer's, Parkinson's, and Huntington's diseases. The nucleating role of amyloid beta (Aβ ) peptide into amyloids is well established. Herein, lipid hybrid-vesicles are generated with glycerol/cholesterol-bearing polymers aiming to alter the nucleation process and modulate the early phases of Aβ fibrillation.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results.

A Quantitative and Reliable Calibration Standard for Dual-Color Fluorescence Cross-Correlation Spectroscopy.

Dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS) allows binding analysis of biomolecules. Combining cross- and autocorrelation amplitudes yields binding degrees and concentrations of bound and unbound species. However, non-ideal detection volume overlap reduces the cross-correlation, causing overestimation of the K .

Oligomerisation of Synaptobrevin-2 Studied by Native Mass Spectrometry and Chemical Cross-Linking.

Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins.

A novel sample preparation strategy for shotgun lipidomics of phospholipids employing multilamellar vesicles.

The identification of lipids in biological samples is gaining importance. The advent of mass spectrometry-based lipidomics accelerated the field allowing nowadays for identification and quantification of complete lipidomes. However, due to solubility difficulties and varying properties of different lipid classes, sample preparation for lipidomics is still an issue.

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies.

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques.

Membrane protein reconstitution into liposomes guided by dual-color fluorescence cross-correlation spectroscopy.

Proteoliposomes represent nanoscale assemblies of indispensable value for studying membrane proteins in general and membrane transporters in particular. Since no universal protocol exists, conditions for proteoliposome formation must be determined on a case-by-case basis. This process will be significantly expedited if the size and composition of the assemblies can be analyzed in a single step using only microliters of sample.

Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity.

Local and coupled thermodynamic stability of the two-domain and bifunctional enzyme SlyD from Escherichia coli.

SlyD (sensitive to lysis D) is a protein folding helper enzyme comprising peptidylprolyl isomerase as well as chaperone activities at the respective FKBP and IF domains. Both domains coact concerning the peptidylprolyl isomerase activity on protein substrates. Using various biophysical techniques and NMR spectroscopy, the local and global thermodynamic stability of the variant (1-165) of SlyD from Escherichia coli (SlyD*) was characterized.

Transient enzyme-substrate recognition monitored by real-time NMR.

Slow protein folding processes during which kinetic folding intermediates occur for an extended time can lead to aggregation and dysfunction in living cells. Therefore, protein folding helpers have evolved, which prevent proteins from aggregation and/or speed up folding processes. In this study, we present the structural characterization of a long-living transient folding intermediate of RNase T1 (S54G/P55N) by time-resolved NMR spectroscopy.

Conformational plasticity and dynamics in the generic protein folding catalyst SlyD unraveled by single-molecule FRET.

The relation between conformational dynamics and chemistry in enzyme catalysis recently has received increasing attention. While, in the past, the mechanochemical coupling was mainly attributed to molecular motors, nowadays, it seems that this linkage is far more general. Single-molecule fluorescence methods are perfectly suited to directly evidence conformational flexibility and dynamics.

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide.

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers.

NMR relaxation unravels interdomain crosstalk of the two domain prolyl isomerase and chaperone SlyD.

The dynamics of the two domain prolyl-peptidyl cis/trans isomerase and chaperone SlyD was studied on a ps-to-ns time scale to correlate dynamic changes with the catalytic function. (15)N transversal and longitudinal relaxation rates as well as heteronuclear Overhauser effects were determined at different temperatures for Escherichia coli SlyD (EcSlyD) and for Thermus thermophilus SlyD (TtSlyD). With the well established extended Lipari-Szabo approach, the order parameter, S(2), the internal correlation time, τ(e), the exchange rate, R(ex), of the backbone amide protons, and the overall molecular tumbling time, τ(m), were determined.

Crystal structure determination and functional characterization of the metallochaperone SlyD from Thermus thermophilus.

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain.

NMR solution structure of SlyD from Escherichia coli: spatial separation of prolyl isomerase and chaperone function.

SlyD (sensitive to lysis D) is a putative folding helper from the bacterial cytosol and harbors prolyl isomerase and chaperone activities. We determined the solution NMR structure of a truncated version of SlyD (1-165) from Escherichia coli (SlyD*) that lacks the presumably unstructured C-terminal tail. SlyD* consists of two well-separated domains: the FKBP domain, which harbors the prolyl isomerase activity, and the insert-in-flap (IF) domain, which harbors the chaperone activity.

Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination.

Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan.