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BBA - Proteins and Proteomics (v.1824, #10)

Editorial Board (pp. i).

Modulation of beta-amyloid aggregation by engineering the sequence connecting beta-strand forming domains by Yang Hu; HeQiu Zheng; Baihao Su; Michael Hernandez; Jin Ryoun Kim (pp. 1069-1079).

Aggregation of beta-amyloid (Aβ) into oligomers and fibrils is associated with the pathology of Alzheimer's disease. The major structural characteristics of Aβ fibrils include the presence of β sheet-loop-β sheet conformations. Several lines of study suggested a potentially important role of the Aβ loop forming sequence (referred to as the Aβ linker region) in Aβ aggregation. Effects of mutations in several charged residues within the Aβ linker region on aggregation have been extensively studied. However, little is known about oligomerization effects of sequence variation in other residues within the Aβ linker region. Moreover, modulation effects of the Aβ linker mutants on Aβ aggregation have yet to be characterized. Here, we created and characterized Aβ linker variants containing sequences preferentially found in specific β turn conformations. Our results indicate that a propensity to form oligomers may be changed by local sequence variation in the Aβ linker region without mutating the charged residues. Strikingly, one Aβ linker variant rapidly formed protofibrillar oligomers, which did not convert to fibrillar aggregates in contrast to Aβ aggregating to fibrils under similar incubation conditions. Moreover, our results suggest that molecular forces critical in oligomerization and fibrillization may differ at least for those involved in the linker region. When co-incubated with Aβ, some Aβ linker variants were found to induce accumulation of Aβ oligomers. Our results suggest that engineering of the Aβ linker region as described in this paper may represent a novel approach to control Aβ oligomerization and create Aβ oligomerization modulators.► Mutations in Aβ residues V24–N27 may affect a propensity to form oligomers. ► An Aβ variant with mutations in V24–N27 may form stable protofibrillar oligomers. ► An Aβ variant with mutations in V24–N27 can cause accumulation of Aβ oligomers.

Keywords: Abbreviations; Aβ; beta-amyloid; AD; Alzheimer's disease; HCD; Aβ hydrophobic central domain; HPLC; high performance liquid chromatography; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; 5-FAM; 5-carboxyfluorescein; PBSA; phosphate-buffered saline with azide; ThT; thioflavin T; SEC; size exclusion chromatography; FPLC; fast performance liquid chromatography; LMWS; low molecular weight species; CD; circular dichroism; TEM; transmission electron microscopy; PAGE; polyacrylamide gel electrophoresis; SDS; sodium dodecyl sulfateAmyloid; Fibril; Oligomer; Protein–protein interaction; Protein aggregation; Protein design

Exon edited dystrophin rods in the hinge 3 region by Neha Sahni; Khushdeep Mangat; Elisabeth Le Rumeur; Nick Menhart (pp. 1080-1089).

We have studied the properties of a panel of proteins engineered to be end-products of envisioned exon skipping therapy by antisense oligonucleotides, AONs, directed at exon 51 applied to relevant dystrophin defects causing Duchenne muscular dystrophy, DMD. Exon skipping therapy is a leading therapeutic strategy being investigated for the treatment of this devastating genetic disease. AONs targeting exon 51 have progressed furthest in human clinical trials. Exon 51 skipping is applicable to a variety of dystrophin defects found in different patients. Due to the differences in original defect, the end result of the therapy will be different in each case. An open question is whether these differences will produce significant differences in the dystrophin protein so edited. In this study we have identified differences in the stability, structure and lipid binding properties of these end-product proteins produced by exon 51 skipping repair.► Exon skipping repair is a leading therapeutic hope for Duchenne muscular dystrophy. ► Skipping of exon 51 has been tested in boys with various relevant gene defects. ► The final repair is a combination of the original defect and the therapeutic skip. ► We studied 4 different such repairs to assess their fidelity to native dystrophin. ► 2 of the 4 show significantly perturbed structure or function.

Keywords: Dystrophin; Exon-skipping; Stability; Lipid; Duchenne muscular dystrophy

Effects of oxidation of lysozyme by hypohalous acids and haloamines on enzymatic activity and aggregation by Petronio Maicon S. Petrônio; Valdecir F. Ximenes (pp. 1090-1096).

Hen egg white lysozyme (HEL), an antibacterial enzyme, is a prototype protein for studying the physical and chemical events that underlie the formation of amyloid fibril aggregates. Here, we studied alterations in enzymatic activity and aggregation provoked by oxidation of HEL by hypochlorous acid (HOCl), hypobromous acid (HOBr), taurine chloramine (Tau–NHCl), taurine monobromamine (Tau–NHBr), and taurine dibromamine (Tau–NBr₂). Addition of only 4-fold molar excess of Tau–NHBr or Tau–NBr₂ to HEL caused complete depletion of its intrinsic fluorescence, whereas HOCl and HOBr caused 40%–50% bleaching. Tau–NHCl was unable to oxidize lysozyme. The selective effect of bromamines on tryptophan residues had a direct effect on enzymatic activity; bromamines were about two-fold more effective as inhibitors of lysozyme than the acid precursors. The oxidation of HEL by HOCl and HOBr was more effective regarding the aggregation of the protein, which was evidenced by increased turbidity, Rayleigh scattering, and anisotropy. The aggregates presented spectroscopic properties that suggested the formation of amyloid fibrils, as measured by the thioflavin assay. In conclusion, the capacity of Tau–NHBr and Tau–NBr₂ as inhibitors of the bactericidal activity of HEL could represent a role in the exacerbation of pulmonary infection, since leukocytes are rich sources of both taurine and HOBr. Moreover, the oxidation of HEL by just a small excess of hypohalous acids, a condition that could be found in inflammatory sites, may represent a new pathway for initiation of aggregation.► Taurine bromamine and dibromamine are oxidants of tryptophan residues in lysozyme. ► Taurine bromamine and dibromamine are potent inhibitors of lysozyme activity. ► Oxidation of lysozyme by hypohalous acids induces its aggregation. ► Aggregates have properties that suggest the formation of amyloid fibrils.

Keywords: Amyloid fibril; Lysozyme activity; Hypobromous acid; Hypochlorous acid; Taurine monobromamine; Taurine dibromamine

Use of SANS and biophysical techniques to reveal subtle conformational differences between native apo-calmodulin and its unfolded states by Gabriel Gibrat; Liliane Assairi; Constantin T. Craescu; Gaston Hui Bon Hoa; Damarys Loew; Bérangère Lombard; Laura Blouquit; Marie-Claire Bellissent-Funel (pp. 1097-1106).

Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85°C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin.► Heat denaturation of apo-calmodulin is studied by SANS and biophysical techniques. ► At 85°C the protein adopts a polymer conformation with residual local structures. ► The refolded state of apo-calmodulin is different from the native one because of dehydration and deamidation of the heated protein as evidenced by mass spectrometry.

Keywords: Calmodulin; Heat denaturation; Small angle neutron scattering; Circular dichroism; Fluorescence and UV absorbance; Mass spectrometry

Nutriproteomics: A promising tool to link diet and diseases in nutritional research by Vijayalakshmi Ganesh; Navam S. Hettiarachchy (pp. 1107-1117).

Nutriproteomics is a nascent research arena, exploiting the dynamics of proteomic tools to characterize molecular and cellular changes in protein expression and function on a global level as well as judging the interaction of proteins with food nutrients. As nutrients are present in complex mixtures, the bioavailability and functions of each nutrient can be influenced by the presence of other nutrients/compounds and interactions. The first half of this review focuses on the techniques used as nutriproteomic tools for identification, quantification, characterization and analyses of proteins including, two-dimensional polyacrylamide electrophoresis, chromatography, mass spectrometry, microarray and other emerging technologies involving visual proteomics. The second half narrates the potential of nutriproteomics in medical and nutritional research for revolutionizing biomarker and drug development, nutraceutical discovery, biological process modeling, preclinical nutrition linking diet and diseases and structuring ways to a personalized nutrition. Though several challenges such as protein dynamics, analytical complexity, cost and resolution still exist, the scope of applying proteomics to nutrition is rapidly expanding and promising as more holistic strategies are emerging.► Nutriproteomics is the study of interaction of dietary nutrients with the proteome. ► Proteomic techniques involve quantitative and functional approaches. ► Mass spectrometry-based proteomics serves as a powerful tool in protein profiling. ► Biomarker, drug and nutraceutical discoveries are major applications of the field. ► Discussion of challenges vs promises of nutriproteomics in nutrition research

Keywords: Nutriproteomics; Protein; Nutrition; Nutraceutical; Drug development; Mass spectrometry

Structure, dynamics and domain organization of the repeat protein Cin1 from the apple scab fungus by Carl H. Mesarich; Michael Schmitz; Pierre Tremouilhac; Duncan J. McGillivray; Matthew D. Templeton; Andrew J. Dingley (pp. 1118-1128).

Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 ( cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix–loop–helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A¹⁵N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain–domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain–domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in α-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a “beads-on-a-string” organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands.Display Omitted► Structural information of secreted tandem-repeat proteins (STRPs) is sparse. ► We have solved the NMR structure of the first two domains of Cin1, a fungal STRP. ► The repeat domains of Cin1 display a novel three-helix bundle fold. ► NMR dynamic analysis of this construct revealed reciprocal domain–domain mobility. ► The eight domains of full-length Cin1 adopt a dynamic, loosely-packed arrangement.

Keywords: Abbreviations; Cin1; cellophane-induced 1; Cin1-FL; full-length Cin1 protein; Cin1-D1; first domain construct of Cin1; Cin1-D1D2; domains 1 and 2 construct of Cin1; HLH; helix–loop–helix; STRP; secreted tandem-repeat protein; TPR; tetratrico peptide repeats; WGS; whole genome sequencingSecreted repeat-domain protein; NMR; SAXS; Protein dynamics; α-helical repeats; Venturia inaequalis

Spatial distribution of cytoplasmic domains of the Mg²⁺-transporter MgtE, in a solution lacking Mg²⁺, revealed by paramagnetic relaxation enhancement by Shunsuke Imai; Tatsuro Maruyama; Masanori Osawa; Motoyuki Hattori; Ryuichiro Ishitani; Osamu Nureki; Ichio Shimada (pp. 1129-1135).

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.► The orientation of the N and CBS domains of apo MgtE in solution was analyzed by PRE. ► These domains were suggested not fixed in a particular orientation in solution. ► Their spatial distribution was visualized as the atomic probability density. ► Structural mechanism for the efficient capture of Mg²⁺ was suggested.

Keywords: Abbreviations; PRE; paramagnetic relaxation enhancement; TM; transmembrane; MgtE; _CP; cytoplasmic region of MgtE (residues 1–259); HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MTSL; S; -(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate; TROSY; transverse relaxation optimized spectroscopy; Γ; ₂; paramagnetic transverse relaxation enhancement rates; Γ; ₂; ^obs; experimentally observed; Γ; ₂; Γ; ₂; ^crystal; Γ; ₂; calculated from the crystal structure of MgtEMgtE; NMR; Paramagnetic relaxation enhancement; Ensemble simulated annealing; Spatial distribution

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus by Giovanna Cacciapuoti; Francesca Fuccio; Luigi Petraccone; Pompea Del Vecchio; Marina Porcelli (pp. 1136-1143).

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.► We investigated the role of disulfide bonds in stability and folding of SsMTAPII. ► We performed the spectroscopic characterization of SsMTAPII and its mutants. ► We carried out thermal and GdmCl-induced unfolding/refolding experiments. ► Chemical and thermal unfolding are reversible only under reducing conditions. ► Unfolding occurs with dissociation of the native hexamer into denatured monomers.

Keywords: Abbreviations; MTAP; 5′-deoxy-5′-methylthioadenosine phosphorylase; SsMTAPII; 5′-deoxy-5′-methylthioadenosine phosphorylase II from; Sulfolobus solfataricus; PNP; purine nucleoside phosphorylase; MTA; 5′-deoxy-5′-methylthioadenosine; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD; circular dichroism; [θ]; molar ellipticity per mean residue; GdmCl; guanidinium chloride; PIPES buffer; piperazine-N,N′-bis(ethanesulfonic acid); TCEP; Tris-(2-carboxyethyl) phosphine5′-deoxy-5′-methylthioadenosine phosphorylase II from; Sulfolobus solfataricus; Hyperthermophilic protein; Reversible chemical and thermal unfolding; Conformational stability; Disulfide bond

Analysis of amino acid contributions to protein solubility using short peptide tags fused to a simplified BPTI variant by Mohammad Monirul Islam; Monsur A. Khan; Yutaka Kuroda (pp. 1144-1150).

Protein solubility is usually characterized in terms of a hydrophobicity scale, which refers to the free energy of transfer of a molecule from an aqueous to a nonpolar solution and is not a “solubility propensity scale” per se. Using a “host–guest” approach, we measured the effects of short poly-amino-acid tags (guests) on the solubility of a host protein, a simplified bovine pancreatic trypsin inhibitor (BPTI), to which they were fused at the C-terminus. We analyzed 10 amino acid types, representing the full range of biophysical properties (acidic, basic, polar, and hydrophobic). As anticipated, positively charged residues significantly increased the solubility of the model protein, at both pH 4.7 and 7.7, whereas very hydrophobic poly-Ile markedly reduced the solubility of BPTI. Poly-Asp and poly-Glu barely affected BPTI solubility at pH 4.7, but induced an eight to ten-fold increase at pH 7.7, attributable to the ionization of their side chains. Although Pro is the most soluble amino acid, poly-Pro did not affect the protein's solubility. The effects of the other tags on BPTI solubility ranged from none to an eight-fold increase. To ensure that the measured solubility values were context independent and could provide a “solubility propensity scale”, we confirmed that the tags remained independent of the structure, thermal stability, and biochemical activity of the host protein. These observations suggest that this approach is valuable for measuring the solubility propensities of amino acids, which could eventually allow the calculation of a polypeptide's relative solubility from its amino acid sequence.► Hydrophobicity is a measure for aqueous to non polar solution transfer. ► Here we report a novel proper “solubility propensity scale”. ► The solubility propensity was measured for 10 amino acid types. ► This could enable an accurate calculation of a peptide’s solubility.

Keywords: Abbreviations; Simplified BPTI; A single-disulfide-bonded bovine pancreatic trypsin inhibitor variant whose sequence was simplified by multiple alanine replacement; BPTI-19; simplified BPTI containing 19 alanines out of 58 residues; Hydropathy; Kyte Doolittle hydropathy scale; Hydrophobicity; dislike of water in a general senseProtein aggregation; Solubility propensity scale; Thermodynamics; Protein stability; Polypeptide

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BBA - Proteins and Proteomics (v.1824, #10)