ProLuCID, a new algorithm for peptide identification using tandem mass spectrometry and protein sequence databases has been developed. This algorithm uses a three tier scoring scheme. First, a binomial probability is used as a preliminary scoring scheme to select candidate peptides. The binomial probability scores generated by ProLuCID minimize molecular weight bias and are independent of database size. A modified cross-correlation score is calculated for each candidate peptide identified by the binomial probability. This cross-correlation scoring function models the isotopic distributions of fragment ions of candidate peptides which ultimately results in higher sensitivity and specificity than that obtained with the SEQUEST XCorr. Finally, ProLuCID uses the distribution of XCorr values for all of the selected candidate peptides to compute a Z score for the peptide hit with the highest XCorr. The ProLuCID Z score combines the discriminative power of XCorr and DeltaCN, the standard parameters for assessing the quality of the peptide identification using SEQUEST, and displays significant improvement in specificity over ProLuCID XCorr alone. ProLuCID is also able to take advantage of high resolution MS/MS spectra leading to further improvements in specificity when compared to low resolution tandem MS data. A comparison of filtered data searched with SEQUEST and ProLuCID using the same false discovery rate as estimated by a target-decoy database strategy, shows that ProLuCID was able to identify as many as 25% more proteins than SEQUEST. ProLuCID is implemented in Java and can be easily installed on a single computer or a computer cluster.

The extracellular matrix (ECM) is a complex and dynamic meshwork of cross-linked proteins that supports cell polarization and functions and tissue organization and homeostasis. Over the past few decades, mass-spectrometry-based proteomics has emerged as the method of choice to characterize the composition of the ECM of normal and diseased tissues. Here, we present a new release of MatrisomeDB, a searchable collection of curated proteomic data from 17 studies on the ECM of 15 different normal tissue types, six cancer types (different grades of breast cancers, colorectal cancer, melanoma, and insulinoma) and other diseases including vascular defects and lung and liver fibroses. MatrisomeDB (http://www.pepchem.org/matrisomedb) was built by retrieving raw mass spectrometry data files and reprocessing them using the same search parameters and criteria to allow for a more direct comparison between the different studies. The present release of MatrisomeDB includes 847 human and 791 mouse ECM proteoforms and over 350 000 human and 600 000 mouse ECM-derived peptide-to-spectrum matches. For each query, a hierarchically-clustered tissue distribution map, a peptide coverage map, and a list of post-translational modifications identified, are generated. MatrisomeDB is the most complete collection of ECM proteomic data to date and allows the building of a comprehensive ECM atlas.

The proteins Sac7d and Sso7d belong to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius and S. solfactaricus, respectively. These proteins are extremely stable to heat, acid and chemical agents. Sac7d binds to DNA without any particular sequence preference and thereby increases its melting temperature by approximately 40 degrees C. We have now solved and refined the crystal structure of Sac7d in complex with two DNA sequences to high resolution. The structures are examples of a nonspecific DNA-binding protein bound to DNA, and reveal that Sac7d binds in the minor groove, causing a sharp kinking of the DNA helix that is more marked than that induced by any sequence-specific DNA-binding proteins. The kink results from the intercalation of specific hydrophobic side chains of Sac7d into the DNA structure, but without causing any significant distortion of the protein structure relative to the uncomplexed protein in solution.

The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the ␤A-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four ␤-strands (␤A-␤B-␤D-␤C) and two helices (␣2 and ␣3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C 70 polymer rather than the C 55 polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C 30 intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C 55 product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C 55 product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k cat values. W75A showed an 8-fold increase of the FPP K m value, and 22-33-fold increases in the IPP K m values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release. Prenyltransferases catalyze consecutive condensation reactions of isopentenyl pyrophosphate (IPP)1 with allylic pyrophosphate to generate linear isoprenyl polymers. The isoprenylates undergo further modification to form a variety of isoprenoid structures including steroids, terpenes, the side chains of respiratory quinones, carotenoids, natural rubber, the glycosyl carrier lipid, and prenyl proteins (1, 2). E-and Z-type prenyltransferases synthesize trans and cis double bonds, respectively, through the condensation reactions of IPP (3). Each of the E-type enzymes catalyzes the formation of a product having a specific chain length ranging from C 10 to C 50 (4).Two conserved DDXXD motifs are observed in E-type enzymes (5-7). X-ray structural (8) and site-directed mutagenesis studies (9 -12) of farnesyl-pyrophosphate synthase (FPPs) have shown that the first aspartate-rich motif binds the allylic substrate, whereas the second DDXXD binds IPP via Mg 2ϩ . Mutagenesis studies indicate that the 5th amino acid residue (Phe-77) upstream from the first DDXXD plays a critical role in controlling the chain length of the final product formed in the reaction catalyzed by E-type geranylgeranyl-pyrophosphate synthase from archaebacterium (13). By substituting this large residue with the smaller Ser, product synthesis was shifted from the production of C 20 product to C 25 and C 30 products (14). Double (F77...

Sso7d and Sac7d are two small (approximately 7,000 Mr), but abundant, chromosomal proteins from the hyperthermophilic archaeabacteria Sulfolobus solfataricus and S. acidocaldarius respectively. These proteins have high thermal, acid and chemical stability. They bind DNA without marked sequence preference and increase the Tm of DNA by approximately 40 degrees C. Sso7d in complex with GTAATTAC and GCGT(iU)CGC + GCGAACGC was crystallized in different crystal lattices and the crystal structures were solved at high resolution. Sso7d binds in the minor groove of DNA and causes a single-step sharp kink in DNA (approximately 60 degrees) by the intercalation of the hydrophobic side chains of Val 26 and Met 29. The intercalation sites are different in the two complexes. Observations of this novel DNA binding mode in three independent crystal lattices indicate that it is not a function of crystal packing.