ABSTRACT
Bacterial expression of full length beta-amyloid (Abeta) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semisynthetic method in which Abeta1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Abeta1-29-Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Abeta1-29-MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Abeta1-29-MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH2-A30C-Abeta30-40, which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Abeta1-29-MESNA. Native chemical ligation proceeds rapidly and efficiently (>90% yield) to give A30C-Abeta1-40. The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (>90% yield) to give native sequence Abeta1-40. Overall, this system is highly efficient, and can yield approximately 8-10 mg of pure Abeta1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Abeta peptides. We have also expressed an Abeta construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Abeta1-29-Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15N or 13C labels for NMR. Future work will also include extending this system to longer Abeta peptides, such as Abeta1-42.
Subject(s)
Amyloid beta-Peptides/biosynthesis , Amyloid beta-Peptides/chemical synthesis , Peptides/chemical synthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Chitin/chemistry , Chitin/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Peptides/chemistry , Peptides/genetics , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
X-ray diffraction of sphingomyelin-dihydrocholesterol (SM-DChol) monolayers revealed short-ranged ( approximately 25 A) 2D ordering. These nanoclusters show two distinct regions: below the cusp point of the phase diagram (35 mol% DChol), a constant d spacing was observed; above the cusp, the d spacing increases linearly with DChol in accordance to Vegard's law for binary alloys. The components in this lipidic alloy are thus a 65ratio35 SM-DChol entity and excess DChol. Reflectivity data further support the emergence above the cusp of an uncomplexed DChol population with greater vertical mobility.
Subject(s)
Cholestanol/chemistry , Nanostructures/chemistry , Sphingomyelins/chemistry , Membrane Fluidity , Membranes, Artificial , X-Ray DiffractionABSTRACT
[reaction: see text] An efficient synthesis of the C20-C32 segment of the phorboxazoles has been achieved using an enantioselective hetero Diels-Alder reaction catalyzed by Jacobsen's Cr(III) amino indanol Schiff base catalyst.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Oxazoles/chemical synthesis , Catalysis , Chromium/chemistry , Heterocyclic Compounds, 4 or More Rings/chemistry , Indicators and Reagents , Molecular Structure , Oxazoles/chemistry , Schiff Bases/chemistryABSTRACT
The C1-C17 bis-oxane subunit 22 of phorboxazole B is efficiently synthesized by exploiting differential reactivities between similar substituents on the hydropyran rings in 4. Selective dihydroxylation of the equatorial vinyl group, hydroboration of the axial vinyl group, and intramolecular Mitsunobu lactonization serve to fully differentiate the similar hydropyrans.
