A new dataset of 396 protein domains is developed and used to evaluate the performance of the protein secondary structure prediction algorithms DSC, PHD, NNSSP, and PREDATOR. The maximum theoretical Q3 accuracy for combination of these methods is shown to be 78%. A simple consensus prediction on the 396 domains, with automatically generated multiple sequence alignments gives an average Q3 prediction accuracy of 72.9%. This is a 1% improvement over PHD, which was the best single method evaluated. Segment Overlap Accuracy (SOV) is 75.4% for the consensus method on the 396-protein set. The secondary structure definition method DSSP defines 8 states, but these are reduced by most authors to 3 for prediction. Application of the different published 8- to 3-state reduction methods shows variation of over 3% on apparent prediction accuracy. This suggests that care should be taken to compare methods by the same reduction method. Two new sequence datasets (CB513 and CB251) are derived which are suitable for cross-validation of secondary structure prediction methods without artifacts due to internal homology. A fully automatic World Wide Web service that predicts protein secondary structure by a combination of methods is available via http://barton.ebi.ac.uk/.

/pdf/evaluation-and-improvement-of-multiple-sequence-methods-for-292rc0zndi.pdf

Evaluation and Improvement of Multiple Sequence Methods for Protein Secondary Structure Prediction

Hydrolysis and amino acid composition analysis of proteins

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80°C and 5, respectively; and the Km was 374 mM, with a Vmax of 1.72 mmol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50°C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.

/pdf/purification-to-homogeneity-and-characterization-of-a-novel-3c2jug2dyb.pdf

Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase.

Most advances in biology can usually be traced back to the development of a new technique: the recent explosion in sequence information in the databases arose from the pioneering work on separation methods by Frederick Sanger which paved the way for the development of protein (Sanger, 1945) and DNA/RNA (Maxam & Gilbert, 1977; Sanger, 1981) sequencing and culminated in the receipt of two Nobel prizes by Sanger. The initial phase of sequence database expansion was slow due to the tedious and slow nature of protein sequencing. Peptide sequencing was carried out manually and the complete analysis of a protein was tiresome, requiring the isolation of sufficient peptides from several digests of the target protein using proteases of different specialities to collect an overlapping set of fragments which cover the whole sequence. Protein sequencing gained momentum when the phenylisothiocyanate sequencing chemistry developed by Edman in 1949 was automated (Edman & Begg, 1967) and a commercial instrument requiring lower amounts (nanomoles) of sample was put on the market. Further technical advances such as novel valves to deal with small volumes of aggressive chemicals, the introduction of high pressure liquid chromatography (HPLC), and novel supports for sample immobilization, were all combined in the first gas phase sequencers, greatly increasing the sensitivity and allowing automated data collection (Hewick et al. 1981) and analysis. The new instruments with a sensitivity in the low picomole range appeared as rapid advances in DNA technology such as the development of restriction mapping (Danna et al. 1973), cloning (Cohen et al. 1973) and the dideoxynucleotide sequencing chemistry were threatening to make protein chemistry a relic of the past (Malcolm, 1978).

/pdf/protein-identification-in-the-post-genome-era-the-rapid-rise-3d7x8ibjus.pdf

Protein identification in the post-genome era: the rapid rise of proteomics

PXylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50°C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-P-D-xylopyranoside as substrate, the K, and V,,,,, values have been found to be 1 .l mM and 25.6 wrnol min- ’ (mg protein)- ‘, respectivdy. Enzyme activity was inhibited by Hg’+, Ag2+, and CL?+ at a concentration of 1 X lop3 M. The synthesis of /3-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the creA gene product.

from Aspergillus nidulans

Probably the most widely used of techniques for analyzing mixtures of proteins is SDS polyacrylamide gel electrophoresis. In this technique, proteins are reacted with the anionic detergent, sodium dodecylsulfate (SDS, or sodium lauryl sulfate) to form negatively charged complexes. The amount of SDS bound by a protein, and so the charge on the complex, is roughly proportional to its size. Commonly, about 1.4 g SDS is bound per 1 g protein, although there are exceptions to this rule. The proteins are generally denatured and solubilized by their binding of SDS, and the complex forms a prolate elipsoid or rod of a length roughly proportionate to the protein's molecular weight. Thus, proteins of either acidic or basic pI form negatively charged complexes that can be separated on the bases of differences in charges and sizes by electrophoresis through a sieve-like matr ix of polyacrylamide gel.

SDS Polyacrylamide Gel Electrophoresis of Proteins.

There is a variety of chemical reactions known to result in the cleavage of the peptide bond. Some are nonspecific-for instance, 6M hydrochloric acid at 110°C for 24 h hydrolyzes a polypeptide to a mixture of single amino acids. Others show some discrimination, however, as to the precise nature of the amino acid residues around the bond to be broken. Some of these methods are sufficiently specific to be of use, for instance, for generating peptides for primary structure determination. These methods usefully augment those that use proteolytic enzymes (see Chapter 5 ), especially since they tend to act at positions occupied by less common amino acids and so generate large peptides.

Chemical cleavage of proteins.

In SDS polyacrylamide gel electrophoresis, proteins are separated essentially on the basis of their sizes, by the sieving effect of the polyacrylamide gel matrix (see Chapter 6 ). In the absence of SDS, the proteins would still be subject to the sieving effect of the gel matrix, but their charges would vary according to their amino acid content. This is because the charge on a protein at any particular pH is the sum of the charges prevailing on the side chain groups of its constituent amino acid residues, and the free amino and carboxyl groups at its termini (although these are relatively trivial in anything other than a very small peptide). Thus, in an ionic detergent-free gel electrophoretic system, both the molecular size and charge act as bases for effective protein separation. The pH prevailing in such a system might be anything, but is commonly about pH 3. Since the pK ( a ) values of the side chain carboxyl groups of aspartic and glutamic acids are about 3.8 and 4.2, respectively, even these amino acids will contribute little to the negative charge on a protein at this pH. Thus at pH 3, all proteins are likely to be positively charged and to travel towards the cathode in an electric field.

Acetic acid-urea polyacrylamide gel electrophoresis of proteins.

There could be various reasons the protein chemist may want to break a protein of interest into fragments, but foremost among them must be the purposes of peptide mapping, primary structure determination, and preparation of particular fragments for studies relating structure and function. Proteinases (or more specifically endo-proteinases) are commonly used to generate peptides for these purposes, but the number of particularly useful and commercially available proteinases is not great. This is because enzyme specificity is an important consideration-the laboratory worker generally requires good yields of clean, pure peptides, but significantly less than 100% cleavage at some or all sites of action gives rise to a complex mixture of a large number of polypeptides.

Bryan John Smith

Papers

SDS Polyacrylamide Gel Electrophoresis of Proteins.

Chemical cleavage of proteins.

Acetic acid-urea polyacrylamide gel electrophoresis of proteins.

Enzymatic methods for cleaving proteins.

Chemical cleavage of polypeptides.