We report on the surface behavior of a hydrophobic, cationic peptide, [lysine-(leucine)4]4-lysine (KL4), spread at the air/water interface at 25 degrees C and pH 7.2, and its effect at very low molar ratios on the surface properties of the zwitterionic phospholipid 1,2-dipalmitoylphosphatidylcholine (DPPC), and the anionic forms of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and palmitic acid (PA), in various combinations. Surface properties were evaluated by measuring equilibrium spreading pressures (pi(e)) and surface pressure-area isotherms (pi-A) with the Wilhelmy plate technique. Surface phase separation was observed with fluorescence microscopy. KL4 itself forms a single-phase monolayer, stable up to a surface pressure pi of 30 mN/m, and forms an immiscible monolayer mixture with DPPC. No strong interaction was detected between POPG and KL4 in the low pi region, whereas a stable monolayer of the PA/KL4 binary mixture forms, which is attributed to ionic interactions between oppositely charged PA and KL4. KL4 has significant effects on the DPPC/POPG mixture, in that it promotes surface phase separation while also increasing pi(e) and pi(max), and these effects are greatly enhanced in the presence of PA. In the model we have proposed, KL4 facilitates the separation of DPPC-rich and POPG/PA-rich phases to achieve surface refinement. It is these two phases that can fulfill the important lung surfactant functions of high surface pressure stability and efficient spreading.

Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.

The two-dimensional crystallization of streptavidin at functionalized lipid interfaces is one of the best studied model systems for investigating molecular self-assembly processes. This system also provides an opportunity to elucidate the relationship between protein−protein molecular recognition, crystallization solution conditions, and crystal properties such as coherence, space group symmetry, and morphology. A better understanding of these relationships may aid in the design of rational strategies for promoting high-quality protein crystallization and for controlling protein assembly at interfaces in the biomaterials and nanotechnology fields. Here we show that two-dimensional streptavidin crystallization is kinetically controlled and that formation of a single electrostatic interaction at the crystal contact interfaces is a key energetic determinant of the kinetic barriers controlling crystal morphology. Our results also demonstrate that this electrostatic interaction at the streptavidin protein−protein interfaces is responsible for the ionic strength dependence of streptavidin crystallization. Molecular modeling studies of the wild-type crystal that displays C222 symmetry suggested that the side-chain amines of lysine 132 from adjacent proteins interact with each other across the dyad-related crystal contacts. Leucine was substituted at this position (K132L) to remove the need for bridging counterions. Unlike wild-type streptavidin, the K132L mutant crystallizes with rectangular morphology on buffer or on a pure water subphase and analysis of the electron micrographs demonstrates that the crystal retains C222 symmetry in the presence or absence of salt. The kinetic barriers associated with formation of this electrostatic interaction thus underlie the wild-type butterfly crystal morphology.

The carboxyl- and amino-terminal ends of streptavidin are near the site of protein−protein contacts in two-dimensional streptavidin crystals. The role of these C- and N-terminal residues in determining the pH-dependent phase behavior of crystallization has been investigated with site-directed truncation mutants. Commercial streptavidin (consisting primarily of amino acids 14−136) and two recombinant streptavidin forms, spanning residues 13−136 and 13−139, have been crystallized at pH 4−7. The commercial 14−136 protein crystallizes in three distinct lattice symmetries, P1, P2, and C 222, respectively, depending on pH. The 13−136 mutant also crystallizes in three distinct lattices, but with a shifted pH profile that is attributed to the N-terminal residue. The presence of amino acids 137−139 inhibits the growth of crystals with P1 symmetry at low pH. In addition, we observe a solid−solid phase transition in situ from the P2 to the P1 crystal forms for the 13−136 recombinant protein at pH 5.2. We also demonstrate the ability of Brewster angle microscopy to distinguish between different crystal forms if protein monolayer densities are sufficiently different.

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