Polymorphism of Oligomers of a Peptide from β‑Amyloid

This contribution reports solution-phase structural studies of oligomers of a family of peptides derived from the β-amyloid peptide (Aβ). We had previously reported the X-ray crystallographic structures of the oligomers and oligomer assemblies formed in the solid state by a macrocyclic β-sheet peptide containing the Aβ_15–23 nonapeptide. In the current study, we set out to determine its assembly in aqueous solution. In the solid state, macrocyclic β-sheet peptide <b>1</b> assembles to form hydrogen-bonded dimers that further assemble in a sandwich-like fashion to form tetramers through hydrophobic interactions between the faces bearing V₁₈ and F₂₀. In aqueous solution, macrocyclic β-sheet peptide <b>1</b> and homologue <b>2a</b> form hydrogen-bonded dimers that assemble to form tetramers through hydrophobic interactions between the faces bearing L₁₇, F₁₉, and A₂₁. In the solid state, the hydrogen-bonded dimers are antiparallel, and the β-strands are fully aligned, with residues 17–23 of one of the macrocycles aligned with residues 23–17 of the other. In solution, residues 17–23 of the hydrogen-bonded dimers are shifted out of alignment by two residues toward the C-termini. The two hydrogen-bonded dimers are nearly orthogonal in the solid state, while in solution the dimers are only slightly rotated. The differing morphology of the solution-state and solid-state tetramers is significant, because it may provide a glimpse into some of the structural bases for polymorphism among Aβ oligomers in Alzheimer’s disease

Aspheric Solute Ions Modulate Gold Nanoparticle Interactions in an Aqueous Solution: An Optimal Way To Reversibly Concentrate Functionalized Nanoparticles

Nanometer-sized gold particles (AuNPs) are of peculiar interest because their behaviors in an aqueous solution are sensitive to changes in environmental factors including the size and shape of the solute ions. In order to determine these important characteristics, we performed all-atom molecular dynamics simulations on the icosahedral Au₁₄₄ nanoparticles each coated with a homogeneous set of 60 thiolates (4-mercapto­benzoate, pMBA) in eight aqueous solutions having ions of varying sizes and shapes (Na⁺, K⁺, tetramethylamonium cation TMA⁺, tris-ammonium cation TRS⁺, Cl^–, and OH^–). For each solution, we computed the reversible work (potential of mean of force) to bring two nanoparticles together as a function of their separation distance. We found that the behavior of pMBA protected Au₁₄₄ nanoparticles can be readily modulated by tuning their aqueous environmental factors (pH and solute ion combinations). We examined the atomistic details on how the sizes and shapes of solute ions quantitatively factor in the definitive characteristics of nanoparticle–environment and nanoparticle–nanoparticle interactions. We predict that tuning the concentrations of nonspherical composite ions such as TRS⁺ in an aqueous solution of AuNPs be an effective means to modulate the aggregation propensity desired in biomedical and other applications of small charged nanoparticles

Conformation-Dependent Human p52Shc Phosphorylation by Human c‑Src

Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-β-d-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation

Identification of SA-binding site on N-terminus of Rad21.

<p>(<b>A</b>) Schematic illustration shows the N-terminal truncated mutants of Rad21 (1–450 aa) with triple mutations (TM) on middle part of SA-binding motif. The cDNA was cloned into pFlag CMV2 vector. (<b>B</b>) Immunoblotting of SA2 co-IP by Rad21 (1–450 aa) WT and mutants. 293 T cells were transfected with the constructs shown in (A) and IP was performed 40 h after transfection. Deleting the first 80 aa of Rad21 (1–450 aa) TM inhibits the co-IP of SA2. The second and third panels are from the same blot. The third panel was enhanced for better visualization. (<b>C</b>) Clustal format alignment of Rand21 (41–90 aa) by MAFFT L-INS-i (v7.015b). The bottom line shows the conserved amino acids from fission yeast (<i>S</i>. <i>pombe</i>) to human Rad21. Invariant, conserved, and semi-conserved residues are indicated by an asterisk (*), colon (:), and period (.), respectively. (<b>D</b>) Schematic illustration shows the full length Rad21 and the mutations on the two SA-binding motifs located on the NT and MP of Rad21, respectively. The cDNAs of Rad21 WT and mutants with quadruple mutations (QM) on the NT SA-binding motif and/or triple mutations (TM) on the MP SA-binding motif were cloned into pFlag CMV2 vector. (<b>E</b>) Immunoblotting of co-IP of cohesin subunits by Rad21 WT or mutants. The dividing lines indicate that interfering lanes have been spliced out.</p

The Missing Linker: A Dimerization Motif Located within Polyketide Synthase Modules

The dimerization of multimodular polyketide synthases is essential for their function. Motifs that supplement the contacts made by dimeric polyketide synthase enzymes have previously been characterized outside the boundaries of modules, at the N- and C-terminal ends of polyketide synthase subunits. Here we describe a heretofore uncharacterized dimerization motif located within modules. The dimeric state of this dimerization element was elucidated through the 2.6 Å resolution crystal structure of a fragment containing a dimerization element and a ketoreductase. The solution structure of a standalone dimerization element was revealed by nuclear magnetic resonance spectroscopy to be consistent with that of the crystal structure, and its dimerization constant was measured through analytical ultracentrifugation to be ∼20 μM. The dimer buries ∼990 Ų at its interface, and its C-terminal helices rigidly connect to ketoreductase domains to constrain their locations within a module. These structural restraints permitted the construction of a common type of polyketide synthase module

Co-immunoprecipitation of other cohesin subunits by Rad21 with mutations or deletion on MP SA-binding motif.

<p>(<b>A</b>) 293 T cells were transfected with pFlag CMV2 Rad21 WT or mutants and co-immunoprecipitation was performed using whole cell lysate. Cells transfected with empty vector (EV) was used as control. Immunoblotting shows the cohesin core subunits and associating proteins immunoprecipitated by Rad21 WT and mutants. (<b>B-C</b>) 293 T cells were transfected with pCS2MT Rad21 WT or mutants. EV was used as control. Endogenous Rad21 was knocked down with Rad21 3′-UTR siRNA. Scrabbled siRNA was used as control (Ctr). Proteins from chromatin fraction were isolated and used for IP. Immunoblotting shows the chromatin fraction was not contaminated by the soluble fraction (B) and like WT Rad21, mutant Rad21 was found on chromatin and co-immunoprecipitated by cohesin-Smc3 (C). EV: empty vector, WT: wild type, DM: L385A T390A, TM, L385A F389A T390A, Del: del(383–392 aa).</p

The Missing Linker: A Dimerization Motif Located within Polyketide Synthase Modules

The dimerization of multimodular polyketide synthases is essential for their function. Motifs that supplement the contacts made by dimeric polyketide synthase enzymes have previously been characterized outside the boundaries of modules, at the N- and C-terminal ends of polyketide synthase subunits. Here we describe a heretofore uncharacterized dimerization motif located within modules. The dimeric state of this dimerization element was elucidated through the 2.6 Å resolution crystal structure of a fragment containing a dimerization element and a ketoreductase. The solution structure of a standalone dimerization element was revealed by nuclear magnetic resonance spectroscopy to be consistent with that of the crystal structure, and its dimerization constant was measured through analytical ultracentrifugation to be ∼20 μM. The dimer buries ∼990 Ų at its interface, and its C-terminal helices rigidly connect to ketoreductase domains to constrain their locations within a module. These structural restraints permitted the construction of a common type of polyketide synthase module

SA2 interacts with middle part of Rad21.

<p>(<b>A</b>) Schematic illustration of the Rad21 deletion constructs (left panel) and Rad21-SA2 interaction results from (B) (right panel). ++: strong interaction; +: weak interaction; −: no interaction. (<b>B</b>) Rad21 interacts with SA2 through the middle region. His-SA2 (1–1051 aa) was expressed along with Flag-Rad21 WT and deletion mutants and co-purified with Ni-NTA or Flag beads. SA2 (1–1051 aa) co-expressed with Flag tagged PA protein was used as a negative control. (<b>C</b>) Gel filtration chromatogram for the SA2 (1–1051 aa):Rad21 (171–450 aa) complex. SA2 (1–1051 aa) and Rad21 (171–450 aa) formed a stable complex. Inset shows the Coomassie-stained gel of the SA2:Rad21 complex purified by gel filtration. (<b>D</b>) Velocity sedimentation results for SA2 (1–1051 aa) and the SA2 (1–1051 aa): Rad21 (171–450 aa) complex. The complex shows an increase in the sedimentation coefficient compared to SA2 alone.</p

SA2 interacts with a 10 aa region of middle part Rad21.

<p>(<b>A</b>) Schematic illustration of the middle portion Rad21 deletion constructs made in the baculovirus system and interaction results of Rad21-SA2 from (B). ++: strong interaction; +: weak interaction; −: no interaction. (<b>B</b>) Rad21 (171–382 aa) does not interact with SA2. His-SA2 (1–1051 aa) was expressed along with the Flag tagged Rad21 deletion mutants and co-purified with Ni-NTA or Flag beads. Antibody cross-reaction bands are marked by asterisk (*). (<b>C</b>) Schematic illustrations of Rad21 deletion constructs in the context of the full length Rad21 in the mammalian expression vector pFlag CMV2 and interaction results from (D). ++: strong interaction; +: weak interaction; −: no interaction. (<b>D</b>) Rad21 383–392 aa region is critical for interacting with SA2. Myc-SA2 (1–1051 aa) was co-transfected along with the Flag-Rad21 deletion mutants and immunoprecipitated with Flag or Myc beads and probed with either the Myc polyclonal antibody (Myc pAb) or the FLAG mAb. Flag empty vector (EV) was used as a negative control.</p