Synthesis of functionalized de novo designed 8-16 kDa model proteins towards metal ion-binding and esterase activity

De novo design and total chemical synthesis of proteins provides a powerful approach for biological and biophysical studies with the ability to prepare artificial proteins with tailored properties, potentially of importance for biophysical studies, material science, nanobioscience, and as molecular probes. In this paper, the previously developed concept of carbohydrates as templates is employed in the de novo design of model proteins (artificial helix bundles) termed 'carboproteins'. The 4-alpha-helix bundle is a macromolecular structure, where four amphiphilic alpha-helical peptide strands form a hydrophobic core. Here this structure is modified towards achieving metal ion-binding and catalytic activity. We report: (i) test of directional effects from different tetravalent carbohydrate templates, (ii) synthesis and evaluation of carboproteins functionalized with phenol, pyridyl or imidazolyl moieties as potential ligands for metal ion-binding as well as for catalysis. Our results include: (i) support of our previous 'controversial' finding that for some carboproteins the degree of alpha-helicity depends on the template, i.e., that there is, to some extent, a controlling effect from the template, (ii) demonstration of binding of Cu(ii) to tetra-functional carboproteins by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), UV-VIS absorption spectroscopy and size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS); (iii) a kinetic investigation of the esterase activity.     

Monolayer assemblies of a de novo designed 4-alpha-helix bundle carboprotein and its sulfur anchor fragment on Au(111) surfaces addressed by voltammetry and in situ scanning tunneling microscopy

Mapping and control of proteins and oligonucleotides on metallic and nonmetallic surfaces are important in many respects. Electrochemical techniques based on single-crystal electrodes and scanning probe microscopies directly in aqueous solution (in situ SPM) have recently opened perspectives for such mapping at a resolution that approaches the single-molecule level. De novo design of model proteins has evolved in parallel and holds promise for testing and controlling protein folding and for new tailored protein structural motifs. In this report we combine these two strategies. We present a scheme for the synthesis of a new 4-alpha-helix bundle carboprotein built on a galactopyranoside derivative with a thiol anchor aglycon suitable for surface immobilization on gold. The carboprotein with thiol anchor in monomeric and dimeric (disulfide) form, the thiol anchor alone, and a sulfur-free 4-alpha-helix bundle carboprotein without thiol anchor have been prepared and investigated for comparison. Cyclic and differential pulse voltammetry (DPV) of the proteins show desorption peaks around -750 mV (SCE), whereas the thiol anchor desorption peak is at -685 mV. The peaks are by far the highest for thiol monomeric 4-alpha-helix bundle carboprotein and the thiol anchor. This pattern is supported by capacitance data. The DPV and capacitance data for the thiolated 4-alpha-helix bundle carboproteins and the thiol anchor hold a strong Faradaic reductive desorption component as supported by X-ray photoelectron spectroscopy. The desorption peak of the sulfur-free 4-alpha-helix bundle carboprotein, however, also points to a capacitive component. In situ scanning tunneling microscopy (in situ STM) of the thiol anchor discloses an adlayer with small domains and single molecules ordered in pin-striped supramolecular structures. In situ STM of thiolated 4-alpha-helix bundle carboprotein monomer shows a dense monolayer in a broad potential range on the positive side of the desorption potential. The coverage decreases close to this potential and single-molecule structures become apparent. The in situ STM contrast is also strengthened, indicative of a new redox-based tunneling mechanism. The data overall suggest that single-molecule mapping of natural and synthetic proteins on well-characterized surfaces by electrochemistry and in situ STM is within reach.     

Monolayers of a de novo designed 4-alpha-helix bundle carboprotein and partial structures on Au(111)-surfaces

Mapping of structure and function of proteins adsorbed on solid surfaces is important in many contexts. Electrochemical techniques based on single-crystal metal surfaces and in situ scanning probe microscopies (SPM) have recently opened new perspectives for mapping at the single-molecule level. De novo design of model proteins has evolved in parallel and holds promise for test and control of protein folding and for new tailored protein structural motifs. These two strategies are combined in the present report.We present a synthetic scheme for a new 4-alpha-helix bundle carboprotein built on a galactopyranoside derivative with a thiol anchor aglycon suitable for surface immobilization on gold. The galactopyranoside with thiol anchor and the thiol anchor alone were prepared for comparison. Voltammetry of the three molecules on Au(111) showed reductive desorption peaks caused by monolayer adsorption via thiolate-Au bonding. In situ STM of the thiol anchor disclosed an ordered adlayer with clear domains and molecular features. This holds promise, broadly for single-molecule voltammetry and the SPM and scanning tunnelling microscopy (STM) of natural and synthetic proteins.     

Hierarchical self-assembly of designed 2 x 2-alpha-helix bundle proteins on Au(111) surfaces

Self-assembled monolayers of biomolecules on atomically planar surfaces offer the prospect of complex combinations of controlled properties, e.g., for bioelectronics. We have prepared a novel hemi-4-alpha-helix bundle protein by attaching two alpha-helical peptides to a cyclo-dithiothreitol (cyclo-DTT) template. The protein was de novo designed to self-assemble in solution to form a 4-alpha-helix bundle, whereas the disulfide moiety enables the formation of a self-assembled monolayer on a Au(111) surface by opening of the disulfide, thus giving rise to a two-step self-assembly process. The 2 x 2-alpha-helix bundle protein and its template were studied by X-ray photo electron spectroscopy (XPS), electrochemical methods, and electrochemical in situ scanning tunneling microscopy (in situ STM). XPS showed that the cyclo-DTT opens on adsorption to a gold surface with the integrity of the 2 x 2-alpha-helix bundle proteins retained. The surface properties of the DTT and 2 x 2-alpha-helix bundle protein adlayer were characterized by interfacial capacitance and impedance techniques. Reductive desorption was used to determine the coverage of the adlayers, giving values of 65 and 16 muC cm(-2) for DTT and 2 x 2-helix, respectively. The 2 x 2-alpha-helix bundle protein adlayers were imaged by in situ STM. The images indicated a dense monolayer according with the voltammetric data. No long-range order could be detected, but two clearly distinct STM contrasts were assigned to 2 x 2-alpha-helix bundle protein molecules oriented in parallel and antiparallel conformations. The template molecule DTT alone forms highly ordered 30-40 nm domains, giving an adlayer density which agreed well with the coverage determined by voltammetry. This could be exploited in STM imaging of mixed DTT/2 x 2-alpha-helix bundle protein monolayers, with clearly distinct STM patterns of the two components.     

De novo design and characterization of copper centers in synthetic four-helix-bundle proteins.

The design and chemical synthesis of de novo metalloproteins on cellulose membranes with the structure of an antiparallel four-helix bundle is described. All possible combinations of three different sets of amphiphilic helices were assembled on cyclic peptide templates which were bound by a cleavable linker to the cellulose. In the hydrophobic interior, the four-helix bundle proteins carry a cysteine and several histidines at various positions for copper ligation. This approach was used successfully to synthesize, for the first time, copper proteins based on a four-helix bundle. UV-vis spectra monitored on the solid support showed ligation of copper(II) by about one-third out of the 96 synthesized proteins and tetrahedral complexes of cobalt(II) by most of these proteins. Three of the most stable copper-binding proteins were synthesized in solution and their structural properties analyzed by spectroscopic methods. Circular dichroism, one-dimensional NMR, and size-exclusion chromatography indicate a folding into a compact state containing a high degree of secondary structure with a reasonably ordered hydrophobic core. They displayed UV-vis absorption, resonance Raman, and EPR spectra intermediate between those of type 1 and type 2 copper centers. The present approach provides a sound basis for further optimizing the copper binding and its functional properties by using combinatorial protein chemistry guided by rational principles.     

Random coils, beta-sheet ribbons, and alpha-helical fibers: one peptide adopting three different secondary structures at will

To potentially cure neurodegenerative diseases, we need to understand on a molecular level what triggers the complex folding mechanisms and shifts the equilibrium from functional to pathological isoforms of proteins. The development of small peptide models that can serve as tools for such studies is of paramount importance. We describe the de novo design and characterization of an alpha-helical coiled coil based model peptide that contains structural elements of both alpha-helical folding and beta-sheet formation. Three distinct secondary structures can be induced at will by adjustment of pH or concentration. Low concentrations at pH 4.0 yield globular particles of the unfolded peptide, while at the same pH, but at higher concentration, defined beta-sheet ribbons are formed. In contrast, at high concentrations and pH 7.4, the peptide forms highly ordered alpha-helical fibers. Thus, this system allows one to systematically study now the consequences of the interplay between peptide and protein primary structure and environmental factors for peptide and protein folding on a molecular level.     

Membrane binding and structure of de novo designed alpha-helical cationic coiled-coil-forming peptides.

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.     

A triply templated artificial beta-sheet.

This paper describes the design, synthesis, and structural evaluation of a compound (4) comprising three molecular templates and a peptide strand that mimics a three-stranded protein beta-sheet. Two of the templates mimic the hydrogen-bonding functionality of peptide beta-strands and serve as the top and bottom strands by embracing the peptide strand, which is located in the middle of the sheet. The remaining template holds the three strands next to each other. The synthesis of artificial beta-sheet 4 begins with the bottom template and involves the sequential addition of the middle and top strands. (1)H NMR chemical shift and NOE studies establish that this compound folds to adopt a hydrogen-bonded beta-sheetlike structure in CDCl(3) solution. Chemical shift studies indicate that triply stranded artificial beta-sheet 4 is more tightly folded than its smaller doubly stranded homologue, artificial beta-sheet 1.     

Temperature profiling of polypeptides in reversed-phase liquid chromatography. II. Monitoring of folding and stability of two-stranded alpha-helical coiled-coils

The present study extends the utility of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor folding and stability of de novo designed synthetic two-stranded alpha-helical coiled-coils. Thus, we have compared the effect of temperature on the RP-HPLC retention behaviour of both oxidized (two identical five-heptad alpha-helical peptides linked by a disulfide bridge) and reduced coiled-coil analogues with various amino acids substituted into the hydrophobic core of the coiled-coil. We were able to correlate the RP-HPLC retention behaviour of the oxidized analogues over the temperature range of 10 to 80 degrees C with the stability of the analogues as determined by conventional thermal and chemical denaturation approaches. In addition, the contribution of a disulfide bridge to coiled-coil stability was highlighted by comparing the elution behaviour of the oxidized and reduced analogues. Overall, we demonstrate the excellent potential of "temperature profiling" by RP-HPLC to monitor differences in oligomerization state and protein stability.     

De novo and inverse folding predictions of protein structure and dynamics

Summary In the last two years, the use of simplified models has facilitated major progress in the globular protein folding problem, viz., the prediction of the three-dimensional (3D) structure of a globular protein from its amino acid sequence. A number of groups have addressed the inverse folding problem where one examines the compatibility of a given sequence with a given (and already determined) structure. A comparison of extant inverse protein-folding algorithms is presented, and methodologies for identifying sequences likely to adopt identical folding topologies, even when they lack sequence homology, are described. Extension to produce structural templates or fingerprints from idealized structures is discussed, and for eight-membered β-barrel proteins, it is shown that idealized fingerprints constructed from simple topology diagrams can correctly identify sequences having the appropriate topology. Furthermore, this inverse folding algorithm is generalized to predict elements of supersecondary structure including β-hairpins, helical hairpins and α/β/α fragments. Then, we describe a very high coordination number lattice model that can predict the 3D structure of a number of globular proteins de novo; i.e. using just the amino acid sequence. Applications to sequences designed by DeGrado and co-workers [Biophys. J., 61 (1992) A265] predict folding intermediates, native states and relative stabilities in accord with experiment. The methodology has also been applied to the four-helix bundle designed by Richardson and co-workers [Science, 249 (1990) 884] and a redesigned monomeric version of a naturally occurring four-helix dimer, rop. Based on comparison to the rop dimer, the simulations predict conformations with rms values of 3–4 ? from native. Furthermore, the de novo algorithms can asses the stability of the folds predicted from the inverse algorithm, while the inverse folding algorithms can assess the quality of the de novo models. Thus, the synergism of the de novo and inverse folding algorthhm approaches provides a set of complementary tools that will facilitate further progress on the protein-folding problem.     

Template-directed assembly of a de novo designed protein

Many naturally occurring biomaterials are composed of laminated structures in which layers of beta-sheet proteins alternate with layers of inorganic mineral. These ordered laminates often have structural and mechanical properties that differ significantly from those of nonbiological materials. An important step in the construction of novel biomaterials is the creation of composites wherein a de novo designed protein assembles into an ordered structure. To achieve this goal, we layered a de novo protein onto the surface of highly ordered pyrolytic graphite (HOPG). The protein was derived from a combinatorial library of novel sequences designed to fold into amphiphilic beta-sheet structures. Atomic force microscopy reveals that the protein assembles on the HOPG surface into ordered fibers aligned in three orientations at 120 degrees to each other. The symmetry and extent of the ordered regions indicate that the hexagonal lattice underlying the graphite surface templates assembly of millions of protein molecules into a highly ordered structure.     

Molecular imprinting on amphiphilic folded polymers for selective molecular recognition in water

We report amphiphilic folded polymers with imprinted nanocavities for selective molecular recognition in water. For this, a molecular imprinting technique is applied to the polymer synthesis: amphiphilic polymer micelles interacting with template molecules are crosslinked in water to fix the folded architecture and memorize the template structure within the polymers; the removal of the templates provides imprint polymers bearing template-specific nanospaces. Here, a hydrophilic dye bearing two anionic groups, Orange G (OG), is used as a model template. For the imprinting, we design amphiphilic random copolymers bearing hydrophilic poly(ethylene glycol) (PEG) chains, hydrophobic olefin groups, and quaternary ammonium groups that can interact with the template. The copolymers were prepared by living radical polymerization and post functionalization. In the presence of OG and methyl blue (MB), the imprinted nanocavity polymers simultaneously capture both of the dyes in water. The total number of encapsulated dyes increased with increasing the number of polymer-bound quaternary ammonium groups. The selectivity of OG against MB increased with the crosslinking density, while imprint polymers encapsulated OG more efficiently than nonimprint polymers. © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020 , 58, 215–224     

Solvent and lipid accessibility prediction as a basis for model quality assessment in soluble and membrane proteins

On-going efforts to improve protein structure prediction stimulate the development of scoring functions and methods for model quality assessment (MQA) that can be used to rank and select the best protein models for further refinement. In this work, sequence-based prediction of relative solvent accessibility (RSA) is employed as a basis for a simple MQA method for soluble proteins, and subsequently extended to the much less explored case of (alpha-helical) membrane proteins. In analogy to soluble proteins, the level of exposure to the lipid of amino acid residues in transmembrane (TM) domains is captured in terms of the relative lipid accessibility (RLA), which is predicted from sequence using low-complexity Support Vector Regression models. On an independent set of 23 TM proteins, the new SVR-based predictor yields correlation coefficient (CC) of 0.56 between the predicted and observed RLA profiles, as opposed to CC of 0.13 for a baseline predictor that utilizes TMLIP2H empirical lipophilicity scale (with standard deviations of about 0.15). A simple MQA approach is then defined by ranking models of membrane proteins in terms of consistency between predicted and observed RLA profiles, as a measure of similarity to the native structure. The new method does not require a set of decoy models to optimize parameters, circumventing current limitations in this regard. Several different sets of models, including those generated by fragment based folding simulations, and decoys obtained by swapping TM helices to mimic errors in template based assignment, are used to assess the new approach. Predicted RLA profiles can be used to successfully discriminate near native models from non-native decoys in most cases, significantly improving the separation of correct and incorrectly folded models compared to a simple baseline approach that utilizes TMLIP2H. As suggested by the robust performance of a simple MQA method for soluble proteins that utilizes more accurate RSA predictions, further significant improvements are likely to be achieved. The steady growth in the number of resolved membrane protein structures is expected to yield enhanced RLA predictions, facilitating further efforts to improve de novo and template based prediction of membrane protein structure.     

Effects of amino acid phi,psi propensities and secondary structure interactions in modulating H alpha chemical shifts in peptide and protein beta-sheet.

H alpha chemical shifts are often used as indicators of secondary structure formation in protein structural analysis and peptide folding studies. On the basis of NMR analysis of model beta-sheet and alpha-helical peptides, together with a statistical analysis of protein structures for which NMR data are available, we show that although the gross pattern of H alpha chemical shifts reflects backbone torsion angles, longer range effects from distant amino acids are the dominant factor determining experimental chemical shifts in beta-sheets of peptides and proteins. These show context-dependent variations that aid structural assignment and highlight anomalous shifts that may be of structural significance and provide insights into beta-sheet stability.     

Approaches to Synthetic Vaccines Design of Epitope-Containing Amphiphilic Peptides Matching the Antigenic Structure in the Native Protein

A general procedure for the design of synthetic vaccines with the retained conformational features of protein antigenic determinants is described. This new concept emerges from detailed studies on the relationship between primary sequence and secondary structure formation of synthetic peptides and takes advantage of the amphiphilic nature of epitope-containing peptide segments in the native protein to accomplish structural modifications. These segments, for example amphiphilic helices or β-sheets, are stabilized by the insertion of secondary structure-inducing amino-acid residues on the hydrophobic part of the peptide without affecting the spatial arrangement of functional residues on the hydrophilic side. The availability of amphiphilic peptides with tailor-made conformational properties, e.g. helices, β-sheets, and, moreover, assemblies of these blocks to structures of higher order (‘folding units’), allows the presentation and stabilization of continuous as well as discontinuous epitopes by this approach. This strategy is exemplified for the case of two discontinuous epitopes taken from lysozyme, which are matched to host molecules with adequate conformational features by the help of computer-assisted molecular modelling. The implications of this new concept for the design of synthetic vaccines are discussed with special emphasis to the important role of peptide synthesis and chemical structure modification.     

全新设计蛋白质的折叠机制

蛋白质全新设计和折叠研究是从两个不同的方向来理解蛋白质序列-结构-功能关系这一结构生物学重要问题. 蛋白质全新设计取得的成功实例一定程度上检验了人们对蛋白质结构和相互作用理解的准确性, 但它们中多数所表现的不同于天然蛋白质的折叠动力学特征也表明, 要达到最终的功能化实现目标还面临着不少的挑战. 本文综述了蛋白质全新设计的发展过程及现状, 蛋白质折叠研究在实验、理论及模拟方面的研究进展, 以及全新设计蛋白质的折叠机制的研究现状. 阐述了深入了解全新设计蛋白质与天然蛋白质折叠机制的不同, 可以为进一步有效地合理化设计蛋白质提供有益的参考.     

A designed glycoprotein analogue of Gc-MAF exhibits native-like phagocytic activity

Rational protein design has been successfully used to create mimics of natural proteins that retain native activity. In the present work, de novo protein engineering is explored to develop a mini-protein analogue of Gc-MAF, a glycoprotein involved in the immune system activation that has shown anticancer activity in mice. Gc-MAF is derived in vivo from vitamin D binding protein (VDBP) via enzymatic processing of its glycosaccharide to leave a single GalNAc residue located on an exposed loop. We used molecular modeling tools in conjunction with structural analysis to splice the glycosylated loop onto a stable three-helix bundle (alpha3W, PDB entry 1LQ7). The resulting 69-residue model peptide, MM1, has been successfully synthesized by solid-phase synthesis both in the aglycosylated and the glycosylated (GalNAc-MM1) form. Circular dichroism spectroscopy confirmed the expected alpha-helical secondary structure. The thermodynamic stability as evaluated from chemical and thermal denaturation is comparable with that of the scaffold protein, alpha3W, indicating that the insertion of the exogenous loop of Gc-MAF did not significantly perturb the overall structure. GalNAc-MM1 retains the macrophage stimulation activity of natural Gc-MAF; in vitro tests show an identical enhancement of Fc-receptor-mediated phagocytosis in primary macrophages. GalNAc-MM1 provides a framework for the development of mutants with increased activity that could be used in place of Gc-MAF as an immunomodulatory agent in therapy.     

Design, folding, and activities of metal-assembled coiled coil proteins

Metal ions serve many purposes in natural proteins, from the stabilization of tertiary structure to the direction of protein folding to crucial roles in electron transfer and catalysis. There is considerable interest in creating metal binding sites in designed proteins to understand the structural role of metal ions and to design new metalloproteins with useful functions. The de novo design of metalloproteins and the role of metals in the folding of designed proteins are reviewed here, with particular focus on the design, folding, and activities of the [M(bpy-peptide)(3)](2+) structure. This maquette is constructed by the covalent attachment of 2,2'-bipyridine to the N-termini of amphiphilic peptides, and it is assembled into a folded trimeric coiled coil by the addition of a six-coordinate transition metal ion and the resulting hydrophobic collapse of the peptides. The [M(bpy-peptide)(3)](2+) structure has been employed in diverse applications, ranging from electron transfer pathway studies to the study of optimal hydrophobic packing in a virtual library to the construction of receptors and biosensors.     

De novo refolding and aggregation of insulin in a nonaqueous environment: an inside out protein remake

While thermodynamic penalties associated with protein-water interactions are the key driving force of folding, perturbed hydration of destabilized protein molecules may trigger aggregation, which in vivo often causes cellular and histological damage. Here we show, that the denatured state of an alpha-helical protein, insulin, converts to a non-native beta-sheet-rich structure upon de novo "refolding" in an anhydrous environment. The beta-pleated conformer precipitates from solutions of DMSO-denatured insulin upon dilution with chloroform. DMSO destroys hydrogen bond network of the native protein acting as a strong acceptor of main chain hydrogen bonds. Upon the addition of chloroform, which is a weak hydrogen bond donor per se, competitive hydrogen bonds between DMSO and chloroform are formed. This leads to the release of unfolded insulin molecules. In the absence of water, the imminent saturation of polypeptide's dandling hydrogen bonds does not produce the native and predominantly alpha-helical state but a beta-sheet-rich structure, which is morphologically and spectrally distinct from insulin amyloid fibrils. Unlike insulin fibrils, the beta-sheet conformer is metastable and refolds spontaneously to the native form in an aqueous environment. This implies that "folding" in the absence of water results in inefficient burial of hydrophobic side-chains, and thermodynamic frustration at the water-protein interface.     

Prediction of Protein Structure Using Surface Accessibility Data

An approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance‐to‐surface information encoded in the sPRE data in the chemical shift‐based CS‐Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach.