Domains as functional building blocks of plant proteins

Emerging evidence in eukaryotic systems suggests that many proteins of diverse cellular processes are made up of protein domains that are well defined in both sequence and structure. This article updates the identification of many ‘classic’ eukaryotic protein domains in various plant cellular processes, with particular emphasis on the non-catalytic categories. We discuss the importance of domains to plant-protein functions and cellular networking, and the emergence of plant-specific domains.     

Assembly of protein building blocks using a short synthetic peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces.     

A 3D building blocks approach to analyzing and predicting structure of proteins

A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of C alpha coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set of standard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can "replace" about 76% of all hexamers in well-refined known proteins with an error of less than 1 A, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.     

Exons – original building blocks of proteins?

In a recent paper, Walter Gilbert's group has estimated the number of original exons from which all extant proteins might have been constructed. The approach used is subjected to a critical analysis here. It is shown that there are flawed assumptions about both the mechanism and generality of exon-shuffling and in the sequence comparison procedures employed, the latter failing to distinguish chance similarity from similarity due to common ancestry. These methodological errors lead to the omission of many known cases of exon-shuffling and the inclusion of others which may not be genuine. In consequence, the analysis from the Gilbert group cannot give a reliable estimate of those modules that actually participated in exon-shuffling and provides no information on the number of protein archetypes that did not participate in these processes.     

Structure by design: from single proteins and their building blocks to nanostructures

Nanotechnology realizes the advantages of naturally occurring biological macromolecules and their building-block nature for design. Frequently, assembly starts with the choice of a "good" molecule that is synthetically optimized towards the desired shape. By contrast, we propose starting with a pre-specified nanostructure shape, selecting candidate protein building blocks from a library and mapping them onto the shape and, finally, testing the stability of the construct. Such a shape-based, part-assembly strategy is conceptually similar to protein design through the combinatorial assembly of building blocks. If the conformational preferences of the building blocks are retained and their interactions are favorable, the nanostructure will be stable. The richness of the conformations, shapes and chemistries of the protein building blocks suggests a broad range of potential applications; at the same time, it also highlights their complexity. In this Opinion article, we focus on the first step: validating such a strategy against experimental data.     

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives

(S-2-amino-5-(aminooxy)pentanoic acid (L -homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L -homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Site-specific cross-linking of proteins through tyrosine hexahistidine tags

The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H(6) tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.     

Utilization of some non coded amino acids as isosters of peptide building blocks

Summary In our study on non coded amino acids and their utilization in peptide chemistry we synthesized methylene-thio (CH₂—S) and methyleneoxy (CH₂—O) group containing amino acids and pseudodipeptides which could be used as building blocks for the construction of peptide hormone analogues. The (CH₂—S) isoster of peptide bond exhibits increased flexibility, lipophility and resistance to proteolytic enzymes. This group exhibits similar properties as the isosteric disulfide bond in the side chain of cystine residue. The (CH₂—O) isoster is moreover similar in its geometry to extended conformation of peptide bond. As a consequence, the changed profile of biological activities could be expected for peptide hormone analogues containing such isosteric moiety. The (CH₂—S) isosters of the peptide bond were prepared by alkylation of thiolates of 2-mercaptocarboxylic acids, the disulfide bond by alkylation of cysteine or homocysteine. The (CH₂—O) isosters were prepared by (AcO)₄Rh₂ catalyzed addition of carbenes of alkyl diazocarboxylates to N-protected aminoalcohols. Pseudodipeptides H—Leu—(CH₂—S)—Gly—NH₂ and H—Leu—(CH₂—O)—Gly—NH₂ were introduced into the C-terminal part of the oxytocin molecule using solution methods of peptide chemistry. Both inserted isosteric bonds were resistant against proteolytic degradation, the first one was found to decrease an enzymic cleavage of the distant Tyr²-Ile³ bond in the corresponding analogue, too. The (CH₂—S) isosters of the disulfide bond containing an orthogonal protection of their-amino (Fmoc) and-(OAll, OH) or-(OBu⁺, OH) carboxylic groups were applied in the solid phase synthesis of the aminoterminal 1-deamino-15-pentadecapeptide of endothelin-I which represents a strong vasoactive agent. The solid phase synthesis was carried out by the step-wise protocol on the Rink or Merrifield type resin using orthogonally protected carba cystine building blocks.     

Mass spectrometric detection of targeting peptide bioconjugation to Red clover necrotic mosaic virus

Plant virus nanoparticle (PVN) formulations constructed from Red clover necrotic mosaic virus by drug infusion and targeting peptide conjugation can be employed as drug delivery tools. In this investigation, we studied the cross-linked structures formed by application of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) and succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester (SMPEG) as heterobifunctional linkers in the bioconjugation process. The plant virus formulations using several targeting peptides cross-linked to the plant virus capsid were characterized by LC/MS(E) analysis, which produced at least 69% sequence coverage using trypsin and chymotrypsin digestion. The results showed evidence for several types of modification located in three domains of the capsid protein. Extensive linker modifications on lysines or cysteines were detected in all the domains, including both intended peptide-capsid cross-links and unintended intracapsid cross-links. Surprisingly, the most extensive peptide modification was observed in the R domain, which is thought to be quite inaccessible to peptides and cross-linking reagents in solution, since it is on the interior of the virus. These results show that heterobifunctional linkers may not be the most efficient method for attachment of peptides to plant virus capsids. As an alternative conjugation strategy, maleimide peptides were used to conjugate with the virus in a one-step reaction. Analysis by LC/MS(E) showed that these one-step maleimide coupling reactions were more specific, such as modifications of C154 and to a lesser extent C267, and provide a means for achieving more effective PVN formulations.     

Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

Periodic distributions of hydrophobic amino acids allows the definition of fundamental building blocks to align distantly related proteins

Several studies on large and small families of proteins proved in a general manner that hydrophobic amino acids are globally conserved even if they are subjected to high rate substitution. Statistical analysis of amino acids evolution within blocks of hydrophobic amino acids detected in sequences suggests their usage as a basic structural pattern to align pairs of proteins of less than 25% sequence identity, with no need of knowing their 3D structure. The authors present a new global alignment method and an automatic tool for Proteins with HYdrophobic Blocks ALignment (PHYBAL) based on the combinatorics of overlapping hydrophobic blocks. Two substitution matrices modeling a different selective pressure inside and outside hydrophobic blocks are constructed, the Inside Hydrophobic Blocks Matrix and the Outside Hydrophobic Blocks Matrix, and a 4D space of gap values is explored. PHYBAL performance is evaluated against Needleman and Wunsch algorithm run with Blosum 30, Blosum 45, Blosum 62, Gonnet, HSDM, PAM250, Johnson and Remote Homo matrices. PHYBAL behavior is analyzed on eight randomly selected pairs of proteins of >30% sequence identity that cover a large spectrum of structural properties. It is also validated on two large datasets, the 127 pairs of the Domingues dataset with >30% sequence identity, and 181 pairs issued from BAliBASE 2.0 and ranked by percentage of identity from 7 to 25%. Results confirm the importance of considering substitution matrices modeling hydrophobic contexts and a 4D space of gap values in aligning distantly related proteins. Two new notions of local and global stability are defined to assess the robustness of an alignment algorithm and the accuracy of PHYBAL. A new notion, the SAD-coefficient, to assess the difficulty of structural alignment is also introduced. PHYBAL has been compared with Hydrophobic Cluster Analysis and HMMSUM methods.     

Site-selective installation of an electrophilic handle on proteins for bioconjugation

Site-selective protein modification strategies can be used to insert non-natural functional groups into protein structures. Herein, we report on the use of the bis-electrophile 3-bromo-2-bromomethyl-1-propene as a reagent to introduce an electrophilic handle at cysteine residues under mild conditions. This method is demonstrated on a variety of proteins containing a solvent-exposed cysteine residue, including an anti-HER2 nanobody. Chemically distinct protein conjugates are then efficiently formed through further reaction of the electrophilic site with various nucleophiles, including thiols and amines. The resulting chemically-defined conjugates are highly stable in the presence of glutathione or human plasma and retain both the structure and function of the native protein.     

Solid-phase route to Fmoc-protected cationic amino acid building blocks

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N-backbone cyclic peptide chemistry.

Protected Nalpha-(aminoallyloxycarbonyl) and Nalpha-(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of Nalpha-backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and alpha,beta-unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side-chain, resulting in fully orthogonal trifunctional building units for the solid-phase peptide synthesis of small cyclic analogs of peptide loops (SCAPELs). Nalpha-amino groups of building units were protected by Fmoc, functional side-chains were protected by t-Bu/Boc/Trt and N-alkylamino or N-alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large-scale solid-phase peptide synthesis. These building units have significant advantage in the synthesis of peptido-related drugs.     

Preparation of omega-functionalized eicosane-phosphate building blocks

New 1,20-substituted eicosanes carrying phosphate headgroups and readily derivatizable thiol, maleimido, and activated carboxylic ester moieties were prepared. The C20-backbone of these molecules was assembled by a halopolycarbon homologation from 1,8-dichlorooctane and 1,6-dibromohexane. 20-Mercapto- and 20-maleimido-icosylphosphates were synthesized via omega-bromo di-t-butyl protected icosylphosphate while 20-phosphonooxy-icosanoic acid N-hydroxysuccinimidoyl ester was prepared via omega-bromo dibenzyl protected icosylphosphate in multistep syntheses. These molecules can serve as model compounds for studying binding and structural organization on different surfaces with potential applications in the fields of biosensors.     

The enhancement by pervanadate of tyrosine phosphorylation on prostatic proteins occurs through the inhibition of membrane-associated tyrosine phosphatases

The Na⁺–K⁺ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na⁺–K⁺ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na⁺–K⁺ ATPase activity in the presence of different concentrations of MgATP or Na⁺ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in V_max and an increase in K_m for MgATP; however, K_a value for Na⁺ was decreased. Treatment of sarcolemma with H₂O₂ plus Fe²⁺, an hydroxyl and other radical generating system, increased MDA content but decreased both Na⁺–K⁺ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na⁺–K⁺ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H₂O₂ plus Fe²⁺ was associated with a decrease in V_max and an increase in K_m for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na⁺–K⁺ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na⁺–K⁺ ATPase activity may be due to a decrease in the affinity of substrate in the sarcolemmal membrane.