Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

Phosphorylation as a tool to modulate aggregation propensity and to predict fibril architecture

Despite the importance of post-translational modifications in controlling the solubility and conformational properties of proteins and peptides, precisely how the aggregation propensity of different peptide sequences is modulated by chemical modification remains unclear. Here we have investigated the effect of phosphorylation on the aggregation propensity of a 13-residue synthetic peptide incorporating one or more phosphate groups at seven different sites at various pH values. Fibril formation was shown to be inhibited when a single phosphate group was introduced at all seven locations in the peptide sequence at pH 7.5, when the phosphate group is fully charged. By contrast, when the same peptides were analysed at pH 1.1, when the phosphate is fully protonated, fibrils from all seven peptide sequences form rapidly. At intermediate pH values (pH 3.6) when the phosphate group is mono-anionic, the aggregation propensity of the peptides was found to be highly dependent on the position of the phosphate group in the peptide sequence. Using this information, combined with molecular dynamics (MD) simulations of the peptide sequence, we provide evidence consistent with the peptide forming amyloid fibrils with a class 7 architecture. The results highlight the potential utility of phosphorylation as a method of reversibly controlling the aggregation kinetics of peptide sequences both during and after synthesis. Moreover, by exploiting the ability of the phosphate group to adopt different charge states as a function of pH, and combining experimental insights with atomistic information calculated from MD simulations as pH is varied, we show how the resulting information can be used to predict fibril structures consistent with both datasets, and use these to rationalise their sensitivity of fibrillation kinetics both to the location of the phosphate group and its charge state.     

Electrochemical and Conformational Consequences of Copper (CuI and CuII) Binding to β‐Amyloid(1–40)

Copper‐induced structural rearrangements of Aβ40 structure and its redox properties are described in this study. Electrochemical and fluorescent methods are used to characterise the behaviour of Aβ–Cu species. The data suggest that time‐dependent folding of Aβ–Cu species may cause changes in the redox potentials.

     

A Chemical Approach to Immunoprotein Engineering: Chemoselective Functionalization of Thioester Proteins in Their Native State

Less than 6 feet under : Serum proteins C3, C4, and α₂M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

     

D-Peptide and D-Protein Technology: Recent Advances,Challenges, and Opportunities**

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.     

Role of the Chemical Environment beyond the Coordination Site: Structural Insight into FeIII Protoporphyrin Binding to Cysteine‐Based Heme‐Regulatory Protein Motifs

The importance of heme as a transient regulatory molecule has become a major focus in biochemical research. However, detailed information about the molecular basis of transient heme–protein interactions is still missing. We report an in‐depth structural analysis of Fe^III heme–peptide complexes by a combination of UV/Vis, resonance Raman, and 2D‐NMR spectroscopic methods. The experiments reveal insights both into the coordination to the central iron ion and into the spatial arrangement of the amino acid sequences interacting with protoporphyrin IX. Cysteine‐based peptides display different heme‐binding behavior as a result of the existence of ordered, partially ordered, and disordered conformations in the heme‐unbound state. Thus, the heme‐binding mode is clearly the consequence of the nature and flexibility of the residues surrounding the iron ion coordinating cysteine. Our analysis reveals scenarios for transient binding of heme to heme‐regulatory motifs in proteins and demonstrates that a thorough structural analysis is required to unravel how heme alters the structure and function of a particular protein.     

The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding

The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.     

Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

The cytotoxic complex formed between α‐lactalbumin and oleic acid (OA) has inspired many studies on protein–fatty acid complexes, but structural insight remains sparse. After having used small‐angle X‐ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein–oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core–shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off‐loading of the OA into cell membranes.     

Protein Dynamics: From Molecules,to Interactions,to Biology

Proteins have a remarkably rich diversity of dynamical behaviors, and the articles in this issue of the International Journal of Molecular Sciences are a testament to that fact. From the picosecond motions of single sidechains probed by NMR or fluorescence spectroscopy, to aggregation processes at interfaces that take months, all time scales play a role. Proteins are functional molecules, so by their nature they always interact with their environment. This environment includes water, other biomolecules, or larger cellular structures. In a sense, it also includes the protein molecule itself: proteins are large enough to fold and interact with themselves. These interactions have been honed by evolution to produce behaviors completely different from those of random polymers.     

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show β-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially ¹⁵N:¹³C-labeled samples, that the β-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.     

Synthetic Peptide templates for molecular recognition: recent advances and applications

The creation of molecular systems that can mimic some of the properties of natural macromolecules is one of the major endeavors in contemporary protein chemistry. However, the construction of artificial proteins with predetermined structure and function is difficult on account of complex folding pathways. The use of topological peptide templates has been suggested to induce and stabilize defined secondary and tertiary structures. This is because the recent advances in the chemistry of coupling reagents, protecting groups, and solid-phase synthesis have made the chemical synthesis of peptides with conformationally controlled and complex structures feasible. Besides their use as structure-inducing devices, these peptide templates can also be utilized to construct novel structures with tailor-made functions. Herein, we present recent advances in the field of peptide-template-based approaches with particular emphasis on the demonstrated utility of this approach in molecular recognition, along with related applications.     

High Metal Chelating Properties from Rapeseed Meal Proteins to Counteract Lipid Oxidation in Foods: Controlled Proteolysis and Characterization

Rapeseed meal proteins (RP) are enzymatically hydrolyzed using three individual proteases (Alcalase, Flavourzyme, and Prolyve) and the enzymatic mechanism is studied. Rapeseed hydrolysates are produced under controlled conditions and the Prolyve hydrolysate is separated by membrane filtration. Their capacity to reduce free radicals (by transfer of hydrogen or electron) or transition metals (by electron transfer) in the absence of an oxidizable substrate, their metal chelating capacity as well as the antioxidant performances in model (conjugated autoxidizable triene assay) are investigated. All hydrolysates show a reduction capacity (by transfer of hydrogen or electron) and antioxidant activities, in a dose-dependent manner, which are however not significantly increased in comparison to the native proteins. A noteworthy metal chelating activity of the peptides produced with Prolyve is highlighted. These results indicate the potential of valorization of RP as a source of high metal chelating peptides to counteract lipid oxidation in foods. Practical applications: Over the last decade, the antioxidative potential of peptides from plant biomass has been evidenced by much research. Considering the myriad of possible sources and the diversity of technology and means to obtain peptides from protein materials, it is reasonable to expect more applications. Concomitantly, preventing lipid oxidation, especially with the polyunsaturated fat-based products, is a major concern in sectors such as agri-food and cosmetic. Although the efficacy of synthetic antioxidants is recognized, both consumers and manufacturers are looking for more innovative, healthy, environmental-friendly processes and quality products. In this context, a controlled proteolysis of proteins from plant by-products can be used as a sustainable strategy to produce antioxidant peptides. Among them, new peptides released from rapeseed proteins with Prolyve can provide interesting metal chelators to counteract lipid oxidation in foods.     

HMGA1a protein unfolds or refolds synthetic DNA-chromophore hybrid polymers: a chaperone-like behavior

High group mobility protein, HMGA1a, was found to play a chaperone-like role in the folding or unfolding of hybrid polymers that contained well-defined synthetic chromophores and DNA sequences. The synthetic and biological hybrid polymers folded into hydrophobic chromophoric nanostructures in water, but existed as partially unfolded configurations in pH or salt buffers. The presence of HMGA1a induced unfolding of the hybrid DNA-chromophore polymer in pure water, whereas the protein promoted refolding of the same polymer in various pH or salt buffers. The origin of the chaperone-like properties probably comes from the ability of HMGA1a to reversibly bind both synthetic chromophores and single stranded DNA. The unfolding mechanisms and the binding stoichiometry of protein-hybrid polymers depended on the sequence of the synthetic polymers.