Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.

Probability-based protein identification by searching sequence databases using mass spectrometry data.

http://nhjy.hzau.edu.cn/kech/ssyy/pdf/drought/4.PDF

Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

https://science.sciencemag.org/content/sci/253/5022/872.full.pdf

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein

Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters

ROS signaling: the new wave?

Acetyl-CoA carboxylase (ACCase; EC 6413) is a biotin containing enzyme found in all organisms and represents the first committed step towards fatty acid and flavanoid biosynthesis in higher plants In the dicot Brassica napus , as in all other dicots studied to date, there exist two distinct forms of the enzyme [1, 2] The Type I form has been well characterized in Arabidopsis thalina and B napus and consists of a single polypeptide, some 220-250kDa in length The Type II form has only recently been revealed in higher plants however In contrast to the Type I form, Type II ACCase consists of four separate subunits, Biotin Carboxylase (BC), Biotin Carboxyl Carrier Protein (BCCP), and Carboxyl-Transferase α and β (CT α and β) This multi-subunit structure is consistent with the structure of acetyl-CoA carboxylase in prokaryotes and algae The biotin containing protein of the Type II form has been shown to be located within purified chloroplasts from pea and oilseed rape [2] The genetic organization of these subunits is also unique The gene for CT β is encoded by a single gene located on the chloroplast genome whereas the other subunits are all nuclear encoded and are present as multiple copies in the Brassica genome The genetic and multi-subunit structure of the Type II ACCase points to a complicated mechanism for co-regulation of expression of the three nuclear encoded genes, cross-talk with the chloroplast genome to regulate expression of the CT β subunit, and finally a molecular mechanism to arrange and organize the individual subunits into a complete and functional protein once translated into the chloroplast itself

Acetyl-CoA Carboxylase from Brassica napus

We demonstrate that treatments with NaCI and the non-ionic osmoticum sorbitol cause acidification in the extracellular medium in Arabidopsis thaliana cell suspension cultures, a typical hyperosmotic stress response in plants. We also show that when administered for 96 hours at equi-osmolar concentrations, sorbitol, unlike NaCI, was not lethal to Arabidopsis cells. A combination of [ 35 S]methionine/cysteine radiolabelling, 2-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight (Maldi-Tof) mass spectrometry revealed that the abundance of heat shock protein (hsp) 70.1 increased as a result of both sorbitol and NaCI treatments in Arabidopsis. This novel finding, resulting from the first proteomics analysis of NaCl-and sorbitol-stressed Arabidopsis, is interpreted and discussed in the light of what we know of the osmoprotective role of hsp70.1 in animal and microbial systems.

Proteomic identification of an hsp70.1 protein induced in Arabidopsis cells following hyperosmotic stress treatments

cDNA clones encoding NADPH-linked 3 oxoacyl-ACP reductase (EC 1.1.1.100) were isolated from a Brassica napus (rape) developing seed library using a probe derived from a full length cDNA clone from Arabidopsis thaliana leaf. There is strong sequence homology, both at the nucleotide and amino acid level, for the cDNAs coding for this enzyme from Brassica napus seed and Arabidopsis thaliana leaf. Northern blots analysis was performed using mRNA isolated from rape leaves and seeds, and demonstrated that the expression of this gene in seeds is aproximately 20 times higher than in leaves. Expression of this gene in an appropriate E. coli expression vector resulted in the accumulation of the 3-oxoacyl-ACP reductase protein, which was accompanied by an increase in the enzymatic activity

cDNA cloning and overexpression of 3-oxoacyl-ACP reductase from Brassica napus seed

Identification of the N -terminal blocking group of rat mammary gland acyl fatty acid synthetase-thioesterase II

Phosphatidate phosphatase (EC 3.1.3.4) catalyses the dephosphorylation of phosphatidic acid to diacylglycerol prior to the final acylation step of the Kennedy Pathway. Unlike the acyltransferases, phosphatidate phosphatase (PAP) has received very little attention in plant lipid research. PAP appears to be the rate limiting factor in triacylglycerol biosynthesis, and has been shown to be controlled by translocation to and from the endoplasmic reticulum [1]. As well as in triacylglycerol biosynthesis, both phosphatidic acid (PA) and diacylglycerol (DAG) are intermediates for the major phospholipids. Therefore PAP lies at a theoretical branchpoint between triacylglycerol and phospholipid biosyntheses, the production of these two lipid species may be governed by the extent of translocation of PAP within the cell. It remains to be seen whether PAP plays a role in signal transduction as has been suggested in animal systems, where it plays a pivotal role by regulating the amounts of both lipid second messengers, PA and DAG. It has been found that there are several isoforms of PAP in both yeast and animals, these appear to serve the different functions.

Antoni R. Slabas

Papers

Acetyl-CoA Carboxylase from Brassica napus

Proteomic identification of an hsp70.1 protein induced in Arabidopsis cells following hyperosmotic stress treatments

cDNA cloning and overexpression of 3-oxoacyl-ACP reductase from Brassica napus seed

Identification of the N -terminal blocking group of rat mammary gland acyl fatty acid synthetase-thioesterase II

The Purification and Characterisation of Phosphatidate Phosphatase from Avocado