Inhibition of the Fibrillation of Amyloid Aß1-40 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 (Aß1-40 ) 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 Aß1-40 fibrillation. Hybrid-vesicles (±100 nm) are prepared by incorporating variable amounts of cholesterol-/glycerol-conjugated poly(di(ethylene glycol)m acrylates)n polymers into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes. The in vitro fibrillation kinetics coupled to transmission electron microscopy (TEM) is employed to investigate the role of hybrid-vesicles on Aß1-40 fibrillation without destroying the vesicular membrane. Both polymers, when embedded in hybrid-vesicles (up to 20%) significantly prolonged the fibrillation lag phase (tlag ) compared to a small acceleration in the presence of DOPC vesicles, irrespective of the amount of polymers inside the hybrid-vesicles. Along with this notable retardation effect, a morphological transformation of the amyloid's secondary structures to amorphous aggregates or the absence of fibrillar structures when interacting with the hybrid-vesicles is confirmed by TEM and circular dichroism (CD) spectroscopy.

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. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

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. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies. Here, we employ three Synaptobrevin-2 variants-the full-length protein Syb(1-116), the soluble, cytosolic variant Syb(1-96) as well as a shorter version Syb(49-96) containing structured segments but omitting a trigger site for SNARE complex formation-to study oligomerisation in the absence of interaction partners or when incorporated into the lipid bilayer of liposomes. Combining native mass spectrometry with chemical cross-linking, we find that the truncated versions show increased oligomerisation. Our findings from both techniques agree well and confirm the presence of oligomers in solution while membrane-bound Synaptobrevin-2 is mostly monomeric. Using ion mobility mass spectrometry, we could further show that lower charge states of Syb(49-96) oligomers, which most likely represent solution structures, follow an isotropic growth curve suggesting that they are intrinsically disordered. From a technical point of view, we show that the combination of native ion mobility mass spectrometry with chemical cross-linking is well-suited for the analysis of protein homo-oligomers. Graphical Abstract ᅟ.

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 Kd . The overlap quality factor that relates measured and true cross-correlation amplitudes has been difficult to determine, because neither a perfect 1 : 1 labeled sample nor perfectly overlapping volumes are readily accomplished. Here, we describe how a stochastically labeled sample can be used for quantitative calibration. Lipid vesicles doped with green and red fluorescent dyes yield highly reproducible relative cross-correlations and allow determination of the setup-dependent overlap quality factor. This reliable, affordable and quick-to-prepare calibration standard expedites any quantitative co-localization or binding analysis by dcFCCS.

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. Of the many lipid classes, phospholipids are the major components of biological membranes. In solution, they spontaneously form lipid vesicles of various structures such as liposomes. They are therefore often used as membrane mimics when studying biological membranes and membrane proteins. Here, we present a novel sample preparation strategy for shotgun lipidomics employing liposomes prepared from lipid standards or lipid mixtures allowing the analysis of phospholipids directly from lipid bilayers. We validated our strategy for lipid identification by tandem mass spectrometry in positive or negative ion mode using different phospholipid species from various classes. We further tested our strategy for relative quantification by mixing different ratios of phospholipid species as well as determining the distribution of lipid species in a natural lipid extract. Graphical abstract ᅟ.

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. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement. In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

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. Here we show that dual-color fluorescence cross-correlation spectroscopy (FCCS) is of great value for optimizing the reconstitution process, because it distinguishes micelles, liposomes and aggregates in heterogeneous mixtures and permits direct monitoring of the co-localization of proteins and lipids in the diffusing assemblies. As proof-of-principle, liposomes containing the functional multidrug resistance transporter NorA from Staphylococcus aureus were prepared, demonstrating that FCCS is an excellent tool to guide the development of reconstitution protocols.

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. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis.

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. Structurally, both domains are rather independent. The urea-induced unfolding transitions of the two domain protein monitored by 2D NMR spectroscopy and amide proton exchange experiments, however, showed that the IF domain experiences a reduced local stability under both native and unfolding conditions compared to the FKBP domain. Nevertheless, the entire protein shows highly cooperative unfolding at elevated denaturing conditions. This cooperativity is significantly reduced in a SlyD* variant missing the IF domain. The quite low local stability due to high internal fluctuations of the IF domain might be the prerequisite for the ubiquitous chaperone function of SlyD. One physiological role of the metallochaperone SlyD is divalent cations binding. Nickel binds only to the FKBP domain but extensively increases the thermodynamic stability of both SlyD domains, verifying the coupled stability of the domains.

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. NMR resonances of this kinetic folding intermediate could be assigned mainly by a real-time 3D BEST-HNCA. These assignments were the basis to investigate the interaction sites between the protein folding helper enzyme SlyD(1-165) (SlyD*) from Escherichia coli (E. coli) and this kinetic intermediate at a residue resolution. Thus, we investigated the Michaelis-Menten complex of this enzyme reaction, because the NMR data acquisition was performed during the actual catalysis. The interaction surface of the transient folding intermediate is restricted to a region around the peptidyl-prolyl bond (Y38-P39), whose isomerization is catalyzed by SlyD*. The interaction surface regarding SlyD* extends from specific amino acids of the FKBP domain forming the peptidyl-prolyl cis/trans-isomerase active site to almost the entire IF domain. This illustrates an effective interplay between the two functional domains of SlyD* to facilitate protein folding catalysis.

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. By labeling the enzyme SlyD, a member of peptidyl-prolyl cis-trans isomerases of the FK506 binding protein type with an inserted chaperone domain, with donor and acceptor fluorophores for single-molecule fluorescence resonance energy transfer, we directly monitor conformational flexibility and conformational dynamics between the chaperone domain and the FK506 binding protein domain. We find a broad distribution of distances between the labels with two main maxima, which we attribute to an open conformation and to a closed conformation of the enzyme. Correlation analysis demonstrates that the conformations exchange on a rate in the 100 Hz range. With the aid from Monte Carlo simulations, we show that there must be conformational flexibility beyond the two main conformational states. Interestingly, neither the conformational distribution nor the dynamics is significantly altered upon binding of substrates or other known binding partners. Based on these experimental findings, we propose a model where the conformational dynamics is used to search the conformation enabling the chemical step, which also explains the remarkable substrate promiscuity connected with a high efficiency of this class of peptidyl-prolyl cis-trans isomerases.

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

The oligomerization of Aß 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 Aß and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aß peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aß fibrillation. The three histidine residues at positions 6, 13 and 14 of Aß(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

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. The study was extended to a relaxation analysis of the peptide bound state for both SlyD species. We found highly different relaxation and dynamic behavior of the two domains for free SlyD. Surprisingly, in the presence of a substrate for the chaperone domain, the ps-to-ns dynamics in the remote center of the prolyl-peptidyl cis/trans isomerization domain increases. We observed this crosstalk between the two domains for both EcSlyD and TtSlyD.

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. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing beta-strand, suggesting in turn a mechanism for chaperone activity by 'donor-strand complementation.' Furthermore, we identified a conserved metal (Ni(2+)) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.

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. The IF domain is inserted into a loop of the FKBP domain near the prolyl isomerase active site. The NMR structure of SlyD* showed no distinct orientation of the two domains relative to each other. In the FKBP domain, Tyr68 points into the active site, which might explain the lowered intrinsic prolyl isomerase activity and the much lower FK506 binding affinity of the protein compared with archetype human FKBP12 (human FK506 binding protein with 12 kDa). The thermodynamics and kinetics of substrate binding by SlyD* were quantified by fluorescence resonance energy transfer. NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides. Insulin aggregation is markedly slowed by SlyD* as evidenced by two-dimensional NMR spectroscopy in real time, probably due to SlyD* binding to denatured insulin. The capacity of the IF domain to establish an initial encounter-collision complex, together with the flexible orientation of the two interacting domains, makes SlyD* a very powerful catalyst of protein folding.

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. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.