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Human Cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD lo ) [Baldwin ET, Bhat, T.N., Gulnik, S., Hosur, M.V., Sowder, R.C., Cachau, R.E., Collins, J., Silva, A.M. and Erickson J.W. (1993) Proc. Natl. and an inactive, high pH (pH 7.5) form (CatD hi ) [Lee AY, Gulnik SV, Erickson JW. (1998) Nat Struct Biol 5:866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD hi and CatD lo and that Y10 stabilizes CatD hi [Lee AY, Gulnik SV, Erickson JW. (1998) Nat Struct Biol 5:866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer BM and Dunn BM. (1996) J Biol Chem 271:15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher k cat /K m values (2, 3, and 6-fold, respectively) at pH 3.7 and pH 4.75 compared to srCatD indicating that these residues are important in stabilizing the CatD hi . E5Q exhibits a 2-fold lower k cat /K m compared to srCatD at both pH values indicating the importance of E5 in stabilizing the CatD lo . Accordingly, full time-course "pH-jump" (pH 5.5 to 4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD hi to CatD lo compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD lo to CatD hi and Y10 stabilizes CatD hi by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD lo .Cathepsin D (CatD) is a member of the pepsin family of aspartic peptidases. It has an acidic pH optimum (pH 2.8 to 4.0) (1,2) and was originally detected in the endosomal and lysosomal compartments of higher eukaryotes (3). Traditionally, CatD was believed to play an important role in general proteolysis during bulk turnover at acidic pH (4). However, numerous studies showed that CatD plays other important physiological roles (5) some of which may involve restricted proteolysis. These include parathyroid hormone (6) processing, antigen processing (7)(8), and the generation of angiostatin from plasminogen (9). CatD has also been implicated in pathological events including tumor growth and metastasis in breast and colorectal cancer (10)(11)(12)(13)(14). Consequently, CatD is a valuable target for the design of inhibitors. One of the most pronounced structural differences between CatD lo and CatD hi is the relocation of the N-terminal segment (residues 3-7). This segment constitutes the first interdomain β-strand of a 6-stranded β-sheet that forms the base of the molecule in CatD lo . In CatD hi , however, it is rea...

We demonstrate a scalable continuous feed method for the synthesis of polymer Janus nanoparticles with different composition and with different aspect ratios between lobes, including with one hard cross-linked polystyrene...

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A novel method is demonstrated to encapsulate titanium dioxide pigment using directed polymerization‐induced self‐assembly (PISA) with reversible addition‐fragmentation chain‐transfer (RAFT) controlled emulsion polymerization. The polymerization is carried out in a batch process in which both styrene (Sty) and the pigment are emulsified using triblock amphiphilic macro‐RAFT copolymers as stabilizers. RAFT‐controlled chain growth leads to directed lamellar self‐assembly, forming polystyrene (PS) shells’ encapsulating pigment particles with 100% efficiency. The pigment resides either at centers of single‐void vesicles or within the interior of multivoid vesiculated particles. The presence of complex morphologies such as spherical particles, nanofibers, nanoplatelets, and polymer vesicles confirms the PISA pathway. The process is optimized to preferably produce polymer‐vesiculated pigment for use as an enhanced opacifier in water‐based paint.

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The phase behavior and self-assembly structures of a series of amphiphilic diblock co-oligomers comprising an ionizable hydrophilic block (5 to 10 units of acrylic acid) and a hydrophobic block (5 to 20 units of n-butyl acrylate, t-butyl acrylate, or ethyl acrylate), synthesized by RAFT polymerization, have been examined by polarizing optical microscopy and small-angle X-ray scattering (SAXS). Self-assembled structure and lyotropic phase stability in these systems is highly responsive to the degree of ionization of the acrylic acid hydrophilic block (i.e., pH), concentration, and nature of the hydrophobic block. Increasing headgroup ionization switched the amphiphiles from behaving like soluble to insoluble surfactants. Liquid isotropic (micellar), hexagonal, lamellar, and discrete cubic phases were found under different solution conditions. The surfactant packing parameter was adapted to understand the self-assembly structures in these diblock co-oligomers. The hydrophobic chain structure and length were shown to strongly affect the relative stabilities of these phases, allowing the self-assembled structure to be varied at will.

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