currents
Docking Dilem m a Even as the Protein Database (PDB) fills up with structures of proteins that represent potential drug targets, designing candidate drugs that bind these proteins remains a challenge. No matter how well characterized a protein is, it is still difficult to determine where a ligand might bind. Most docking algorithms start with the assumption that the ligand-binding site has been identified. But to evaluate, de novo, the potential binding sites over the surface of even a small protein is a daunting task for any computer system. To address this problem, Graham Richards and his colleagues at the University of Oxford (U.K.) took a multiscale approach ( J. Am. Chem. Soc. 2002, 124, 2337−2344). Their method relies on the application of a scaling operator that removes the most detailed data, leaving behind only the major features. A k-means-clustering algorithm, which establishes model atom clusters based on the distances and geometries of actual atoms, is then applied to the ligand to generate a series of models, each of which is slightly more complex than the previous iteration. A grid-based method is then used to generate a table of ligand−protein pair interaction energies. At this point, the modeling begins. The first model represents the ligand as a single point on the protein molecular field. Positions that place the point too close or too far from the field are rejected from the list of possible docking configurations. A second model then represents the ligand as two points; this is rotated through the remaining
CellM icroarrays
(Ithaca, NY) Wilson Xu might have solved the problem with the construction of a cell microarray system (Genome Res. 2002, 12, 482−486). Xu looked at a variety of support materials and settled on cellulose ester permeable membranes, which provided the right combination of transparency, inertness, and permeability to drugs and nutrients. Using robot-controlled pins that were programmed to form 100- to 1500-pL nanocraters, Xu arrayed 30-pL cell droplets from cultures grown
DNA and protein microarrays offer researchers a great deal of information about the presence and behavior of these molecules during the development of a cell and how they interact with other cellular constituents. These experiments are limited, however, by the fact that they work in a veritable vacuum, only addressing the behaviors of two or three constituents at a time. To follow cell metabolism, a highthroughput cellular method is required. Cornell University’s Robotic pins Cell droplet Membrane (a)
(b)
(c)
Nanocraterm aker.(a) Robotic pins that carry 30-pL cell culture droplets are lowered toward a support membrane, (b) eventually pressing into the membrane. (c) When the pins are withdrawn, they leave behind an array of nanocraters filled with cells. © 2002 American Chemical Society
Realorm odeled? Taking a multiscale approach, researchers predicted the binding of nevirapine to HIV-reverse transcriptase (red). The predicted position (blue) is very close to that determined by X-ray crystallography (yellow). (Adapted from J. Am. Chem. Soc. 2002,124, 2337−2344.)
configuration possibilities, and unfavorable positions are rejected. This process continues with successively more complex ligand models until only a few possible configurations remain. The researchers tested their method on a number of protein−ligand complexes that they pulled from the PDB, removing the ligand from the protein structure and then seeing if they could accurately model the same structure. With the complex of HIV-reverse transcriptase and its nonnucleotide inhibitor nevirapine (see figure), they were able to predict the binding site and orientation to within 1.15 Å of the crystal structure. By performing further energy minimization calculations, they were able to bring the difference down to 0.32 Å. Other modeled complexes (e.g., streptavidin/biotin, trypsin/benzamidine, and cytochrome P-450/camphor) came similarly close to the PDB structures. The researchers said that the accuracy of their method is limited by both the resolution of the protein grid and the size of the rotation angle, but that it offers an excellent starting point for rapid local optimization.
in a 96-well plate. The cratered membrane was then placed on a nutrient medium for cell growth and testing. Xu first arrayed E. coli cultures that did or did not express β-galactosidase on a medium containing chromogenic substrates of the enzyme. Only those cells that carried the β-galactosidase gene showed a color reaction indicating proper enzyme expression and cell growth, as well as the absence of cross-contamination between the cultures. Xu then arrayed auxotrophic yeast cultures that carried one of the genes LEU2 or TRP1 and grew the cells on a medium containing either leucine or tryptophan. Only the LEU2 cells grew on the leucine-minus medium, and similarly only the TRP1 cells grew on the tryptophanminus medium, indicating
metabolic selectivity. Xu then arrayed yeast cultures that were deficient in one of 94 genes. One strain was deficient in FKB1, a gene that codes for a protein that binds to a cell and makes it sensitive to the antifungal drug rapamycin. Only the cells that were deleted for FKB1 were able to grow on rapamycin-containing media. This result showed that the array system would be useful in screening cells for the effects of various drugs. Xu concluded that the cell microarrays provide a high-throughput screening mechanism, to test the effects of small molecule compounds on cell metabolism, growth, and development, providing more accurate answers to questions in drug development, genomics, and proteomics.
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currents Am plifying ProteinBinding Dom ains Yeast two-hybrid screening is instrumental in the development of protein interaction maps. But mapping protein functional domains, typically involving 100 or so residues, has required either preliminary sequence information or arduous trial-and-error experiments. To simplify this problem, Yasuaki Kawarasaki and colleagues at Nagoya University (Japan) developed a Clone 1 P2
P1 P1
Clone 2 Recombination
PASA-PCR
Dom ain recom bination.When two positive clones are isolated from a two-hybrid screen, they might carry the same protein-interaction domain (blue). After in vitro recombination, the products can be amplified by PASA-PCR, which selectively reproduces the shorter clone. (Adapted with permission from Anal. Biochem. 2002, 303, 34–41. Copyright 2002 Elsevier Science.)
PCR-based method, which they call preferentially amplified short amplicon PCR (PASA-PCR) (Anal. Biochem. 2002, 303, 34–41). In conventional PCR, a primer (P1) is annealed to a plasmid containing a DNA fragment to be amplified and is extended with DNA polymerase until it reaches the other end of the fragment, which is adjacent to another primer-binding site (P2). This second site then acts as a template for the next round of PCR. In PASA-PCR, the annealing/extension time of the first two PCR cycles is limited to 5 s (vs 120 s) so that the
polymerase cannot synthesize DNA beyond a certain length. Thus, only amplicons within a certain size range will be produced. Experimentally, this range was 500−1100 bp. Unfortunately, any clone that gives a positive twohybrid signal typically carries DNA sequences beyond the domain of interest, which means that a large number of clones would need to be sequenced to identify the domain. This problem can be circumvented, however, by in vitro reP2 combination between two clones covering the same sequence (see figure). Because of its size, the shorter recombined fragment, carrying only the domain of interest, will be preferentially amplified. The researchers tested their system on the yeast gene for SPC34, a component of the spindle pole body complex, which plays a key role in cell division. Kawarasaki’s group wanted to define the domain within SPC34 that interacts with SPC19, another member of the complex. They constructed a library of SPC34 DNA fragments and transformed the library into a yeast two-hybrid screen, assaying for interactions with SPC19. Positive clones were pooled and subjected to PASA-PCR. The resulting amplicons were then rescreened, and positive clones were sequenced. Common to each of the positive clones was a sequence that covered the C-terminal half of SPC34, which suggested that this sequence was sufficient for interaction with SPC19. Although this experiment was performed with a selected library (SPC34 sequences only), the researchers are confident of its wide applicability.
Quantitative ESI-M S Protein phosphorylation plays a dominant role in cell signaling pathways. Thus, a thorough understanding of where and when these events occur is critical to an understanding of how cells function. Various methods have been used to identify which amino acids in a protein are phosphorylated, but these methods are largely qualitative. To answer these questions quantitatively, Meredith Bond and colleagues developed a capillary LC/ESIMS system that compares a phosphopeptide of interest to an internal reference peptide (Anal. Chem. 2002, 74, 1658– 1664). Normally, when a reference peptide is used in MS, it is added to the peptide mixture just before MS analysis. Thus, it does not go through the same reaction and purification steps as the native
ratio of the MS peak areas of the peptide of interest and the reference, the researchers quantitatively assess the degree of phosphorylation. This ratio can then be converted to mole of phosphorylation per mole of protein by comparing the peak areas to a calibration curve of synthetic peptide standards. The researchers tested their method by following the phosphorylation kinetics of serine-44 (Ser44) in cardiac troponin I (cTnI). The cTnI was treated with protein kinase C βII and ATP, and aliquots were run on SDSPAGE. The cTnI bands were digested in-gel with trypsin, and the resulting peptides were analyzed by capillary LC/ESI-MS. The researchers concentrated their efforts on the peptide 41ISASR45, where Ser44 is phosphorylated, and the native reference peptide, 121NITEIADLTQK131. As cTnI is
Peak replacem ent.After 5 min of in vitro phosphorylation of cardiac troponin, both 41ISASR45 and 41ISASpR45 (phosphorylated) peptides were present in the trypsin-digested sample. As the reaction proceeded, the latter peak totally replaced the former. (Adapted from Anal. Chem. 2002,74, 1658–1664.)
peptides and so cannot be compared quantitatively. To alleviate this concern, Bond’s group developed the “native reference peptide” method, which relies on a peptide that is present in the protein digest being studied but is unmodified. This also eliminates experimentally introduced variations. By taking a
phosphorylated, the phosphopeptide peak slowly replaces the unphosphorylated peptide peak. This is indicated by changes (in the opposite direction) of the chromatographic peak area ratio of each of the two test peptides to the reference peptide. Although the native reference peptide method was es-
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currents tablished for use with phosphopeptides, the researchers believe that it will be useful for the quantitation of any protein modification.
3-D GelElectrophoresis To address the problems associated with 2-D gel electrophoresis (2DE)poor resolution, the swamping of lowabundance signals, and the inability to identify hydrophobic proteins—researchers can physically divide proteomes into smaller units, often by isolating cell organelles. But even these proteomes can be too complex for accurate resolution by 2DE. Although researchers were able to describe 650−800 2DE spots for the mitochondrial proteome of
M
W
Blue-native
pH
3-D effects. By first isolating and purifying protein complexes from a native gel, researchers can more clearly identify the proteins that form these complexes by 2-D electrophoresis (by pH and molecular weight [MW]).
Arabidopsis thaliana, this number was still only a percentage of the 2000−3000 putative proteins. Thus, Wolf Werhahn and Hans-Peter Braun of the Institut für Angewandte Genetik (Hannover, Germany) proposed the use of a third electrophoretic dimension (Electrophoresis 2002, 23, 640−646). Werhahn and Braun isolated mitochondria and outer membrane fractions from A. thaliana and treated the proteins under native conditions with Coomassie blue, which altered the charge of the proteins without greatly affecting their weight or structure. The proteins were then run on a Blue-native (BN) gel, and bands representing individ206
H/D Exchange and Protein Identification Peptide mass fingerprinting (PMF), which involves mass spectral analysis of peptides resulting from the proteolytic digestion of a protein, is invaluable for protein identification. But where 4−5 peptides and a mass uncertainty of 2−3 Da were once sufficient to identify the parent protein, the rapid growth of the protein databases has increased the stringency requirements for correct identification such that at least 10 peptides with a mass error of
