Two proteins for the price of one: Structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1.

Seed storage proteins are both an important source of nutrition for humans and essential for seedling establishment. Interestingly, unusual napin-type 2S seed storage albumin precursors in sunflowers contain a sequence that is released as a macrocyclic peptide during post-translational processing. The mechanism by which such peptides emerge from linear precursor proteins has received increased attention; however, the structural characterization of intact precursor proteins has been limited. Here, we report the 3D NMR structure of the Helianthus annuus PawS1 (preproalbumin with sunflower trypsin inhibitor-1) and provide new insights into the processing of this remarkable dual-destiny protein. In seeds, PawS1 is matured by asparaginyl endopeptidases (AEPs) into the cyclic peptide SFTI-1 (sunflower trypsin inhibitor-1) and a heterodimeric 2S albumin. The structure of PawS1 revealed that SFTI-1 and the albumin are independently folded into well-defined domains separated by a flexible linker. PawS1 was cleaved in vitro with recombinant sunflower HaAEP1 and in situ using a sunflower seed extract in a way that resembled the expected in vivo cleavages. Recombinant HaAEP1 cleaved PawS1 at multiple positions, and in situ, its flexible linker was removed, yielding fully mature heterodimeric albumin. Liberation and cyclization of SFTI-1, however, was inefficient, suggesting that specific seed conditions or components may be required for in vivo biosynthesis of SFTI-1. In summary, this study has revealed the 3D structure of a macrocyclic precursor protein and provided important mechanistic insights into the maturation of sunflower proalbumins into an albumin and a macrocyclic peptide.

Production of isotope-labeled proteins in insect cells for NMR.

Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host.

Natural structural diversity within a conserved cyclic peptide scaffold.

We recently isolated and described the evolutionary origin of a diverse class of small single-disulfide bonded peptides derived from Preproalbumin with SFTI-1 (PawS1) proteins in the seeds of flowering plants (Asteraceae). The founding member of the PawS derived peptide (PDP) family is the potent trypsin inhibitor SFTI-1 (sunflower trypsin inhibitor-1) from Helianthus annuus, the common sunflower. Here we provide additional structures and describe the structural diversity of this new class of small peptides, derived from solution NMR studies, in detail. We show that although most have a similar backbone framework with a single disulfide bond and in many cases a head-to-tail cyclized backbone, they all have their own characteristics in terms of projections of side-chains, flexibility and physiochemical properties, attributed to the variety of their sequences. Small cyclic and constrained peptides are popular as drug scaffolds in the pharmaceutical industry and our data highlight how amino acid side-chains can fine-tune conformations in these promising peptides.

Diverse cyclic seed peptides in the Mexican zinnia (Zinnia haageana).

A new family of small plant peptides was recently described and found to be widespread throughout the Millereae and Heliantheae tribes of the sunflower family Asteraceae. These peptides originate from the post-translational processing of unusual seed-storage albumin genes, and have been termed PawS-derived peptides (PDPs). The prototypic family member is a 14-residue cyclic peptide with potent trypsin inhibitory activity named SunFlower Trypsin Inhibitor (SFTI-1). In this study we present the features of three new PDPs discovered in the seeds of the sunflower species Zinnia haageana by a combination of de novo transcriptomics and liquid chromatography-mass spectrometry. Two-dimensional solution NMR spectroscopy was used to elucidate their structural characteristics. All three Z. haageana peptides have well-defined folds with a head-to-tail cyclized peptide backbone and a single disulfide bond. Although two possess an anti-parallel ß-sheet structure, like SFTI-1, the Z. haageana peptide PDP-21 has a more irregular backbone structure. Despite structural similarities with SFTI-1, PDP-20 was not able to inhibit trypsin, thus the functional roles of these peptides is yet to be discovered. Defining the structural features of the small cyclic peptides found in the sunflower family will be useful for guiding the exploitation of these peptides as scaffolds for grafting and protein engineering applications.

A tripartite approach identifies the major sunflower seed albumins.

KEY MESSAGE: We have used a combination of genomic, transcriptomic, and proteomic approaches to identify the napin-type albumin genes in sunflower and define their contributions to the seed albumin pool. Seed protein content is determined by the expression of what are typically large gene families. A major class of seed storage proteins is the napin-type, water soluble albumins. In this work we provide a comprehensive analysis of the napin-type albumin content of the common sunflower (Helianthus annuus) by analyzing a draft genome, a transcriptome and performing a proteomic analysis of the seed albumin fraction. We show that although sunflower contains at least 26 genes for napin-type albumins, only 15 of these are present at the mRNA level. We found protein evidence for 11 of these but the albumin content of mature seeds is dominated by the encoded products of just three genes. So despite high genetic redundancy for albumins, only a small sub-set of this gene family contributes to total seed albumin content. The three genes identified as producing the majority of sunflower seed albumin are potential future candidates for manipulation through genetics and breeding.

A chameleonic macrocyclic peptide with drug delivery applications.

Head-to-tail cyclized peptides are intriguing natural products with unusual properties. The PawS-Derived Peptides (PDPs) are ribosomally synthesized as part of precursors for seed storage albumins in species of the daisy family, and are post-translationally excised and cyclized during proteolytic processing. Here we report a PDP twice the typical size and with two disulfide bonds, identified from seeds of Zinnia elegans. In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two ß-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This dimer can be split by addition of micelles or organic solvent and in monomeric form PDP-23 adopts open or closed V-shapes, exposing different levels of hydrophobicity dependent on conditions. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrate this by conjugating a rhodamine dye to PDP-23, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump.

Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system.

Protein purification processes in basic research using ÄKTA™ liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS™ autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applications, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein.

Mature forms of the major seed storage albumins in sunflower: A mass spectrometric approach.

UNLABELLED: Seed storage albumins are abundant, water-soluble proteins that are degraded to provide critical nutrients for the germinating seedling. It has been established that the sunflower albumins encoded by SEED STORAGE ALBUMIN 2 (SESA2), SESA20 and SESA3 are the major components of the albumin-rich fraction of the common sunflower Helianthus annuus. To determine the structure of sunflowers most important albumins we performed a detailed chromatographic and mass spectrometric characterization to assess what post-translational processing they receive prior to deposition in the protein storage vacuole. We found that SESA2 and SESA20 each encode two albumins. The first of the two SESA2 albumins (SESA2-1) exists as a monomer of 116 or 117 residues, differing by a threonine at the C-terminus. The second of the two SESA2 albumins (SESA2-2) is a monomer of 128 residues. SESA20 encodes the albumin SESA20-2, which is a 127-residue monomer, whereas SESA20-1 was not abundant enough to be structurally described. SESA3, which has been partly characterized previously, was found in several forms with methylation of its asparagine residues. In contrast to other dicot albumins, which are generally matured into a heterodimer, all the dominant mature sunflower albumins SESA2, SESA20-2, SESA3 and its post-translationally modified analogue SESA3-a are monomeric. BIOLOGICAL SIGNIFICANCE: Sunflower plants have been bred to thrive in various climate zones making them favored crops to meet the growing worldwide demand by humans for protein. The abundance of seed storage proteins makes them an important source of protein for animal and human nutrition. This study explores the structures of the dominant sunflower napin-type seed storage albumins to understand what structures evolution has favored in the most abundant proteins in sunflower seed.

Proteomic allergen-peptide/protein interaction assay for the identification of human skin sensitizers.

Modification of proteins by skin sensitizers is a pivotal step in T cell mediated allergic contact dermatitis (ACD). In this process small reactive chemicals interact covalently or non-covalently with cellular or extracellular skin self-proteins or self-peptides to become recognized by the human immune system. Aiming to develop a novel non-animal in vitro test system for predicting sensitization potential of small reactive chemicals in human skin the allergen-peptide/protein interaction assay (APIA) has been developed. By applying modern proteomic technologies together with a target peptide containing all amino acids, the assay permits the profiling of all amino acid specific allergen-peptide interactions. Moreover, potentially crucial allergen-specific Cys-modifications are qualitatively monitored by mass spectrometry and confirmed by a dual peptide approach. Assay conditions chosen mimic the distinct human epidermal reactivity compartments of the skin surface (pH 5.5), stratum basale (pH 6.8), and typical physiological conditions (pH 7.4). An extreme as well as a moderate human contact sensitizer produced Cys-specific mass shifts, whereas a skin irritant did not. Our data indicate that MALDI-MS based and skin-related in vitro technology platforms - like the APIA - are promising tools in developing alternative non-animal allergen assays. This will assist in chemical classification and next generation risk assessment strategies, including REACH and experimental immunotoxicology.