Patrick J. B. Edwards

N-terminal Domains of DELLA Proteins Are Intrinsically Unstructured in the Absence of Interaction with GID1/Gibberellic Acid Receptors

The plant growth-repressing DELLA proteins (DELLAs) are known to represent a convergence point in integration of multiple developmental and environmental signals in planta, one of which is hormone gibberellic acid (GA). Binding of the liganded GA receptor (GID1/GA) to the N-terminal domain of DELLAs is required for GA-induced degradation of DELLAs via the ubiquitin-proteasome pathway, thus derepressing plant growth. However, the conformational changes of DELLAs upon binding to GID1/GA, which are the key to understanding the precise mechanism of GID1/GA-mediated degradation of DELLAs, remain unclear. Using biophysical, biochemical, and bioinformatics approaches, we demonstrated for the first time that the unbound N-terminal domains of DELLAs are intrinsically unstructured proteins under physiological conditions. Within the intrinsically disordered N-terminal domain of DELLAs, we have identified several molecular recognition features, sequences known to undergo disorder-to-order transitions upon binding to interacting proteins in intrinsically unstructured proteins. In accordance with the molecular recognition feature analyses, we have observed the binding-induced folding of N-terminal domains of DELLAs upon interaction with AtGID1/GA. Our results also indicate that DELLA proteins can be divided into two subgroups in terms of their molecular compactness and their interactions with monoclonal antibodies.

Structural, Dynamic, and Chemical Characterization of a Novel S-Glycosylated Bacteriocin

Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two β-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two α-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.

Milk protein structure - what can it tell the dairy industry?

This paper describes the possible usefulness of knowledge of the three-dimensional structure of milk proteins to dairy scientists. After a brief introduction of the available methodology and the structures of milk proteins that are already available, the structure of bovine β-lactoglobulin is used to illustrate the possible applications of the structure to understanding the problems to which the protein contributes during milk processing.

The production of soluble and correctly folded recombinant bovine β-lactoglobulin variants A and B in Escherichia coli for NMR studies

The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, post-translation modification and secretion of the proteins involved. In the case of bovine beta-lactoglobulin (BLG), a major cows milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis. In our study we show that when co-expressed with the signal-sequence-less disulfide bond isomerase (Delta ssDsbC) in the dual expression vector, pETDUET-1, both BLG A and BLG B can be reproducibly produced in a soluble form. Expression was carried out in Escherichia coli Origami(DE3), a trxB/gor mutant for thioredoxin- and glutathione reductase, which allows for proper formation of disulfide bonds in the cytoplasm. The protein was purified by anion exchange chromatography followed by salting-out at low pH and size exclusion chromatography. Our expression system is able to consistently produce milligram quantities of correctly folded BLG A and B with no additional amino acid residues at the N-terminus, except for a methionine. (15)N-labeled BLG A and B, prepared and purified using this method, produced HSQC spectra typical of native bovine BLG.

Isolation and Structural Identification of the Anthocyanin Components of Red Kiwifruit

The anthocyanins responsible for the red color of red kiwifruit were extracted in acidified ethanol and isolated by solid phase extraction (SPE) followed by preparative HPLC. Five anthocyanins were obtained and subsequently identified as delphinidin 3-[2-(xylosyl)galactoside], delphinidin 3-galactoside, cyanidin 3-[2-(xylosyl)galactoside], cyanidin 3-galactoside, and cyanidin 3-glucoside by a combination of LC-MS/MS, GC-MS, and 2D NMR. Delphinidin 3-[2-(xylosyl)galactoside] and delphinidin 3-galactoside have not previously been reported in the genus Actinidia.

Structural features of bovine caseinomacropeptide A and B by 1H nuclear magnetic resonance spectroscopy.

Samples of bovine caseinomacropeptide (CMP) were isolated from kappa-casein A and kappa-casein B and fractionated to give aglycosylated CMP A and CMP B and monoglycosylated CMP A. The secondary structures of these three peptides were compared under neutral and acidic (pH 4.2) conditions, using two-dimensional (2D) 1H nuclear magnetic resonance (NMR) spectroscopy. The differences between the spectra at pH 4.2 and 7.0 and the spectra of the aglycosylated and glycosylated CMP A were subtle, indicating little change in backbone conformation with these changes. These results Suggest that differences in the coagulation properties of milks containing either kappa-casein A or kappa-casein B are more likely to be related to factors, such as micelle size or charge, than to structural differences arising from altered backbone conformation of the macropeptide segments of the kappa-caseins.

Synthesis of the antimicrobial S-linked glycopeptide, glycocin F.

The first total synthesis of glycocin F, a uniquely diglycosylated antimicrobial peptide bearing a rare S-linked N-acetylglucosamine (GlcNAc) moiety in addition to an O-linked GlcNAc, has been accomplished using a native chemical ligation strategy. The synthetic and naturally occurring peptides were compared by HPLC, mass spectrometry, NMR and CD spectroscopy, and their stability towards chymotrypsin digestion and antimicrobial activity were measured. This is the first comprehensive structural and functional comparison of a naturally occurring glycocin with an active synthetic analogue.

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp32–Asp215 diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

A picornaviral loop-to-loop replication complex

Abstract Picornaviruses replicate their RNA genomes through a highly conserved mechanism that involves an interaction between the principal viral protease (3Cpro) and the 5′-UTR region of the viral genome. The 3Cpro catalytic site is the target of numerous replication inhibitors. This paper describes the first structural model of a complex between a picornaviral 3Cpro and a region of the 5′-UTR, stem-loop D (SLD). Using human rhinovirus as a model system, we have combined NMR contact information, small-angle X-ray scattering (SAXS) data, and previous mutagenesis results to determine the shape, position and relative orientation of the 3Cpro and SLD components. The results clearly identify a 1:1 binding stoichiometry, with pronounced loops from each molecule providing the key binding determinants for the interaction. Binding between SLD and 3Cpro induces structural changes in the proteolytic active site that is positioned on the opposite side of the protease relative to the RNA/protein interface, suggesting that subtle conformational changes affecting catalytic activity are relayed through the protein.

Rheo-NMR Studies of an Enzymatic Reaction: Evidence of a Shear-Stable Macromolecular System

Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious. Here, a custom-built rheo-NMR cell was used to investigate whether shear flow modifies enzyme action compared with that observed quiescently. Specifically, (1)H NMR was used to follow the kinetics of the liberation of methanol from the methylesterified polysaccharide pectin by pectinmethylesterase enzymes. Two different demethylesterifying enzymes, known to have different action patterns, were used. In all experiments performed, Couette flows with shear rates of up to 1570 s(-1) did not generate detectable differences in the rate of methanol liberation compared to unsheared samples. This study provides evidence for a shear-stable macromolecular system consisting of a largely beta-sheet protein and a polysaccharide, in line with current theoretical predictions, but in contrast to some other experimental work on other proteins.