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

Bryksa, Brian C.; Yada, Rickey Y.

doi:10.1038/s41598-017-16734-2

Cited by 7 publications

(14 citation statements)

References 98 publications

(124 reference statements)

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“…The notion that the disulfide bonds guide membrane specificity [5] and have a minimal role in maintaining structure [33] is a reasonable conclusion given the existence of many saposin-like proteins lacking disulfide bonds, and is further supported by the results presented in this work. Such functional examples are found outside of Eukarya and include: a secretion factor in Vibrio vulnificus (Prokarya) [36], a putative calcium-binding protein in Sulfolobus solfataricus (a hyperthermophile in Archaea) [37], various cyclic bacteriocins [38,39], and other small prokaryotic cyclic peptides [40][41][42].…”

Section: Plos Onesupporting

confidence: 76%

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics

et al. 2020

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The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

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Section: Plos Onesupporting

confidence: 76%

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics

et al. 2020

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“…Spectra were taken at the National High Magnetic Field Lab (Tallahassee, FL, USA) using the 40 mm bore Series Connected Hybrid (SCH) magnet system, currently the highest-field NMR magnet [38]. Two-dimensional 13 C-13 C cross polarization [39] dipolar assisted rotational resonance [40] (CP DARR) spectra were obtained at 36 T, with a 2 mm CPMAS probe tuned to frequencies of 1 H, 13 C, 15 N, with ferroshims, at a temperature of 10 • C, a 100 ms mixing time, and a MAS rate of 24.4 kHz [38].…”

Section: Solid-state Nmrmentioning

confidence: 99%

“…There is experimental evidence that PSI of Solanum tuberosum forms dimers under low pH conditions [13], leading to the hypothesis that the activation of D. capensis D1 PSI requires dimerization at low pH values. To test this hypothesis, the presence of monomeric and dimeric D1 PSI was measured by intact protein mass spectrometry at a range of pH values, from 3 to 8 (Supplementary Figure S5).…”

Section: D1 Psi Is Monomeric In Solution Over a Wide Ph Rangementioning

confidence: 99%

“…Structurally, PSIs are categorized as saposin-like proteins, a protein family whose members have substantial sequence diversity but share a strongly conserved, compact tertiary fold, usually stabilized by three disulfide bonds [10]. Although the S. tuberosum PSI is monomeric in solution [11], it was crystallized as a dimer [9] and appears to oligomerize upon interaction with anionic membranes [12,13]. Membrane permeabilization can induce cytotoxicity and is a common antimicrobial defense.…”

Section: Introductionmentioning

confidence: 99%

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The Droserasin 1 PSI: A Membrane-Interacting Antimicrobial Peptide from the Carnivorous Plant Drosera capensis

et al. 2020

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The Droserasins, aspartic proteases from the carnivorous plant Drosera capensis, contain a 100-residue plant-specific insert (PSI) that is post-translationally cleaved and independently acts as an antimicrobial peptide. PSIs are of interest not only for their inhibition of microbial growth, but also because they modify the size of lipid vesicles and strongly interact with biological membranes. PSIs may therefore be useful for modulating lipid systems in NMR studies of membrane proteins. Here we present the expression and biophysical characterization of the Droserasin 1 PSI (D1 PSI.) This peptide is monomeric in solution and maintains its primarily α -helical secondary structure over a wide range of temperatures and pH values, even under conditions where its three disulfide bonds are reduced. Vesicle fusion assays indicate that the D1 PSI strongly interacts with bacterial and fungal lipids at pH 5 and lower, consistent with the physiological pH of D. capensis mucilage. It binds lipids with a variety of head groups, highlighting its versatility as a potential stabilizer for lipid nanodiscs. Solid-state NMR spectra collected at a field strength of 36 T, using a unique series-connected hybrid magnet, indicate that the peptide is folded and strongly bound to the membrane. Molecular dynamics simulations indicate that the peptide is stable as either a monomer or a dimer in a lipid bilayer. Both the monomer and the dimer allow the passage of water through the membrane, albeit at different rates.

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“…The CD spectra of the untreated and released protein from SCDDS prepared via SCF and SE methods are presented in Figure 11. The CD spectrum of the released protein shows similar maxima and minima to untreated bHb, indicating that it largely retained its confirmation after release [47]. The obtained CD spectra were processed by the CDNN software to determine the fraction content of different secondary structures of the protein.…”

Section: Displacermentioning

confidence: 99%

Myristic Acid Coated Protein Immobilised Mesoporous Silica Particles as pH Induced Oral Delivery System for the Delivery of Biomolecules

2019

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Solid core drug delivery systems (SCDDS) were prepared for the oral delivery of biomolecules using mesoporous silica as core, bovine haemoglobin (bHb) as model drug and supercritical fluid (SCF) processing as encapsulation technique. The use of organic solvents or harsh processing conditions in the development of drug delivery systems for biomolecules can be detrimental for the structural integrity of the molecule. Hence, the coating on protein-immobilised particles was performed via supercritical carbon dioxide (scCO2) processing at a temperature lower than the melting point of myristic acid (MA) to avoid any thermal degradation of bHb. The SCDDS were prepared by bHb immobilisation on mesoporous silica followed by myristic acid (MA) coating at 43 °C and 100 bar in scCO2. bHb-immobilised silica particles were also coated via solvent evaporation (SE) to compare the protein release with scCO2 processed formulations. In both cases, MA coating provided required enteric protection and restricted the bHb release for the first two hours in simulated gastric fluid (SGF). The protein release was immediate upon the change of media to simulated intestinal fluid (SIF), reaching 70% within three hours. The release from SCF processed samples was slower than SE formulations, indicating superior surface coverage of MA on particles in comparison to the SE method. Most importantly, the protein conformation remained unchanged after the release from SCDDS as confirmed by circular dichroism. This study clearly demonstrates that the approach involving protein immobilisation on silica and scCO2 assisted melt-coating method can protect biomolecules from gastric environment and provide the required release of a biologic in intestine without any untoward effects on protein conformation during processing or after release.

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

Cited by 7 publications

References 98 publications

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics

The Droserasin 1 PSI: A Membrane-Interacting Antimicrobial Peptide from the Carnivorous Plant Drosera capensis

Myristic Acid Coated Protein Immobilised Mesoporous Silica Particles as pH Induced Oral Delivery System for the Delivery of Biomolecules

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