The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77–124) and a longer (residues 65–124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding

Protein Structure Insights into the Bilayer Interactions of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease

Abstract Many plant aspartic proteases contain a saposin-like domain whose principal functions are intracellular sorting and host defence. Its structure is characterised by helical segments cross-linked by three highly conserved cystines. The present study on the saposin-like domain of Solanum tuberosum aspartic protease revealed that acidification from inactive to active conditions causes dimerisation and a strand-to-helix secondary structure transition independent of bilayer interaction. Bilayer fusion was shown to occur under reducing conditions yielding a faster shift to larger vesicle sizes relative to native conditions, implying that a lower level structural motif might be bilayer-active. Characterisation of peptide sequences based on the domain’s secondary structural regions showed helix-3 to be active (~4% of the full domain’s activity), and mutation of its sole positively charged residue resulted in loss of activity and disordering of structure. Also, the peptides’ respective circular dichroism spectra suggested that native folding within the full domain is dependent on surrounding structure. Overall, the present study reveals that the aspartic protease saposin-like domain active structure is an open saposin fold dimer whose formation is pH-dependent, and that a bilayer-active motif shared among non-saposin membrane-active proteins including certain plant defence proteins is nested within an overall structure essential for native functionality

Nanotechnology: the word is new but the concept is old. An overview of the science and technology in food and food products at the nanoscale level

Food scientists and technologists are actively engaged in examining and developing nanotechnologies for applications such as novel functional ingredients and nutrient delivery systems, safety testing, packaging, and authenticity/authentication at an ever-increasing pace. However, before these new products/technologies are commercialised, rigorous safety testing and risk/benefit analysis are required to ensure that public and environmental concerns are addressed. This review provides an overview of food nanoscience and technology including a brief history, education, definitions pertaining to policy and regulation, and applications. The most recent findings and advances are emphasised, focussing on bioactives' delivery. In addition, proposed directions in the area of nano-based targeting of pathogens for food safety as well as medical foods are discussed. As food nanoscience and technology has been extensively reviewed in recent years, specific case examples will be limited to those reported within the past year.<br /

In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family

The C_α root-mean-square fluctuations (RMSFs) of the PSI dimer as a function of time.

<p>The C_α RMSFs for helices at pH 3.0 (<b>A</b>) and pH 7.4 (<b>B</b>) were consistent throughout the time course of the simulations, remaining below 5 Å for the helical regions. Fluctuations of up to 10 Å were noted for the flexible linker region, in agreement with the hypothesis that the linker region is intrinsically disordered, providing evidence of PSI dimer secondary structure stability regardless of pH. Colours identify the individual peptide chains within the dimer.</p

Comparison of folded potato PSI to other SAPLIPs.

<p>Structural comparison of the folded potato PSI at pH 4.5 (blue) and pH 7.4 (green), averaged over the last 200 ns of the simulation trajectories, to the crystal structure of barley PSI (PDB ID 1QDM, magenta) and the crystal structure Sap C (PDB ID 2GTG, red). Potato PSI simulated at pH 7.4, simulated with parameters closely resembling the experimental parameters used for both barley PSI and Sap C, exhibited a compact globular structure consisting of a distorted four-α-helix bundle characteristic of other SAPLIPs. Potato PSI simulated at pH 4.5 adopted a compact four-α-helix bundle structure not previously observed for any SAPLIP. The linker regions of potato PSI are omitted for clarity.</p

The backbone RMSD of PSI monomers as a function of time.

<p>Backbone RMSD of the PSI monomer at pH 4.5 (blue line) and pH 7.4 (orange line) are presented. The PSI monomer maintains its overall tertiary structure at both pH 4.5 and pH 7.4 similar to that of the native dimer structure until an abrupt change in RMSD at 230 ns and 198 ns for pH 4.5 and pH 7.4, respectively. At these times, the PSI jackknifes closed and adopts saposin-like fold characteristic of all known SAPLIP members.</p

Contribution of the first twenty PCA eigenvectors to the overall closing motion of the PSI.

<p>The contribution of the first 20 PCA eigenvectors to the closing motion of the PSI at active (<b>A</b>) and inactive (<b>B</b>) pH are presented. The first eigenvectors contribute 78.8% and 74.2% of the overall motions for the pH 4.5 and pH 7.4 simulations, respectively, and correspond to the collapse of helices α1/α2 onto helices α3/α4.</p

The backbone RMSD of the PSI dimer as a function of time.

<p>Backbone root-mean-square deviation (RMSD) of the PSI dimer at pH 3.0 (<b>A</b>) and pH 7.4 (<b>B</b>) indicated little deviation, evidenced by the low RMSD of the PSI backbone atoms for both peptides comprising the PSI homodimer. Colours identify the individual peptide chains within the dimer.</p