Determining appropriate solvent conditions is a crucial first step for carrying out NMR spectroscopy of proteins, but rapid and efficient methods for doing so are currently lacking. Microdrop screening examines a large number of different solvent conditions using very small amounts of protein and minimal labor. Starting from one initial buffer condition, small aliquots of protein solution are combined with an array of solutions in which concentration, pH, buffer type, and added stabilizers are systematically varied. The protein concentration of each microliter-sized test drop ('microdrop') is gradually changed using vapor diffusion, and the solubility of the protein is determined by visual examination. A variety of analytical techniques may be applied to the contents of the microdrops to monitor enzymatic activity, aggregation, ligand binding, and protein folding.
"Microdrop screening was used to detect KIR2DS1 aggregation as described by Lepre and Moore . We tested 23 different buffer conditions with a 24 well tissue culture plate and siliconized glass cover slips. 1 mL of each buffer was pipetted into each reservoir, and then, 2 µL aliquots of protein solution in starting buffer were pipetted onto the glass coverslips. 1 µL of each of reservoir buffer was added to each drop, and the solutions were mixed by gently drawing and expelling the solution in the pipette tip. "
[Show abstract][Hide abstract] ABSTRACT: As a major component of the innate immune system, natural killer cells are responsible for activating the cytolytic killing of certain pathogen-infected or tumor cells. The self-recognition of natural killer cells is achieved in part by the killer cell immunoglobulin-like receptors (KIRs) protein family. In the current study, using a suite of biophysical methods, we investigate the self-association of an activating KIR, KIR2DS1. This KIR is of particular interest because when in the presence of the HLA-Cw6 protein, KIR2DS1 becomes a major risk factor for psoriasis, an autoimmune chronic skin disease. Using circular dichroism spectroscopy, dynamic light scattering, and atomic force microscopy, we reveal that KIR2DS1 self-associates in a well-defined fashion. Our novel results on an activating KIR allow us to suggest a working model for the KIR2DS1- HLA class I molecular mechanism.
PLoS ONE 08/2011; 6(8):e23052. DOI:10.1371/journal.pone.0023052 · 3.23 Impact Factor
"Numerous efforts have been devoted to overcoming the solubility and sample stability issues. For example, extensive buffer screening (Bagby et al. 1997; Lepre and Moore 1998), addition of charged amino acids (Golovanov et al. 2004), or introduction of point mutants (Huang et al. 1996; Ito and Wagner 2004; Sun et al. 1999) have been successfully utilized to increase the solubility of the target proteins. However , these methods are often protein specific, largely based on trial and error, and may not be easily applicable to other systems. "
[Show abstract][Hide abstract] ABSTRACT: Although the rapid progress of NMR technology has significantly expanded the range of NMR-trackable systems, preparation of NMR-suitable samples that are highly soluble and stable remains a bottleneck for studies of many biological systems. The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed unapproachable by solution NMR techniques. In this review, we provide a brief survey of the development and successful applications of the SET strategy in biomolecular NMR.We also comment on the criteria for choosing optimal SETs, such as for differently charged target proteins, and recent new developments on NMR-invisible SETs.
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