The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG. The ratio of the forward-to-backward detected SHG provides a measure of the particle size, suggesting detection limits down to crystallites 100 nm in diameter under low magnification (10x). In addition to being sensitive and highly selective, second-order nonlinear optical imaging of chiral crystals (SONICC) is directly compatibility with virtually all common protein crystallization platforms.
A family of 3-methoxypoly(ethylene glycol)-vinyl ether-1,2-dioleylglycerol (mPEG-VE-DOG) lipopolymer conjugates, designed on the basis of DFT calculations to possess a wide range of proton affinities, was synthesized and tested for their hydrolysis kinetics in neutral and acidic buffers. Extruded ~100 nm liposomes containing these constructs in ≥90 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) produced dispersions that retained their calcein cargo for more than 2 days at pH 7.5, but released the encapsulated contents over a wide range of timescales as a function of the electronic properties of the vinyl ether linkage, the solution pH and the mPEG-VE-DOG composition in the membrane. The in vivo performance of two different 90:10 DOPE:mPEG-VE-DOG compositions was also evaluated for blood circulation time and biodistribution in mice, using 125I-tyraminylinulin as a label. The pharmacokinetic profiles gave a T1/2 of 7 h and 3 h for 90:10 DOPE:ST302 and 90:10 DOPE:ST502, respectively, with the liposomes being cleared predominantly by liver and spleen uptake. The behavior of these DOPE:mPEG-VE-DOG formulations is consistent with their relative rates of vinyl ether hydrolysis, i.e., the more acid-sensitive mPEG-VE-DOG derivatives produce faster leakage rates from DOPE:mPEG-VE-DOG liposomes, but decreased the blood circulation times in mice. These findings suggest that the vinyl ether-based PEG-lipid derivatives are promising agents for stabilizing acid-sensitive DOPE liposomes to produce formulations with a priori control over their pH-responsiveness in vitro. Our data also suggest, however, that the same factors that contribute to enhanced acid-sensitivity of the DOPE:mPEG-VE-DOG dispersions are also likely responsible for their reduced pharmacokinetic profiles.
Structure-based drug design (SBDD) has emerged as a valuable pharmaceutical lead discovery tool, showing potential for accelerating the discovery process, while reducing developmental costs and boosting potencies of the drug that is ultimately selected. SBDD is a iterative, rational, lead compound sculpting process that involves both the synthesis of new derivatives and the evaluation of their binding to the target structure either through computational docking or elucidation of the target structure as a complex with the lead compound. This method heavily relies on the production of high-resolution (< 2Å) three-dimensional structures of the drug target, obtained through X-ray crystallographic analysis, in the presence or absence of the drug candidate. The lack of generalized methods for high quality crystal production is still a major bottleneck in the process of macromolecular crystallization. This review provides a brief introduction to SBDD and describes several macromolecular crystallization strategies, with an emphasis on advances and challenges facing researchers in the field today. Recent trends in the development of more universal macromolecular crystallization techniques, particularly nucleation-based techniques that are applicable to both soluble and integral membrane proteins, are also discussed.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.
Biochemistry Z 0250 Design, Synthesis, and Performance of NTA-Modified Lipids as Templates for Histidine-Tagged Protein Crystallization -[144 refs.]. -(THOMPSON*, D. H.; ZHOU, M.; GREY, J.; KIM, H.-K.; Chem. Lett. 36 (2007) 8, 956-975; Dep. Chem., Purdue Univ., West Lafayette, IN 47907, USA; Eng.) -H. Toeppel 49-274
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.