Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data

Institute of Molecular Biology, University of Oregon, Eugene 97403.
Analytical Biochemistry (Impact Factor: 2.22). 12/1989; 182(2):319-26. DOI: 10.1016/0003-2697(89)90602-7
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Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.

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Available from: Peter H. von Hippel, Oct 09, 2015
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    • "Computed pI indicate the acidic nature of laccases under study, probably due to the presence of higher content of negatively charged amino acids (D, E). Extinction coefficient of protein reflects concentration of cys, trp, tyr residues in a particular protein (Gill and Hippel 1989). EC values of selected bacterial laccases were lower than those of fungal laccases probably due to the low content of aromatic amino acids (F, Y, W) and cysteine residue. "
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    ABSTRACT: Laccases from various sources like higher plants, fungi as well as bacteria are known to have wide range of applications in various industries such as paper, textile and beverage. This enzyme is extensively studied because of its vital role in bioremediation, biosensors and diagnostics. Phylogenetic analysis of retrieved sequences of laccases from selected bacteria and fungi inferred variation in sequences and these laccases were grouped independently. All laccases of selected organisms are acidic showing computed pI \ 7 and hydrophilic in nature. Presence of disulfide bridges was observed in laccases of bacteria Pseudomonas putida, Haemophilus parasuis HPS11 and Endozoicomonas numazuensis as well as in all selected fungal laccases. N-myristoylation and phospho-rylation sites for Protein kinase C and Casein kinase II were found in all selected laccases. Secondary structure prediction revealed dominance of random coils in selected laccases. Validation results of predicted 3D structures of laccase enzymes confirmed that the modeled structures were of good quality. All selected bacterial laccases were intracellular and those of fungal were found to be extra-cellular. These investigations provide functional information about these laccases and may help to design successful experimental work aiming towards isolation and purification of laccases from closely related organisms. Experimental structures of these laccase enzymes are not yet available, so till then this study will provide a platform for knowing the structural organization responsible for functioning of these enzymes.
    07/2015; 4(17). DOI:10.1007/s13721-015-0089-y
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    • "To avoid dialysis during the second purification step, eluted fractions from CM25 were applied to a hydrophobic C18 column and finally purified to homogeneity by reversed-phase, high-performance liquid chromatography (RP-HPLC) on a Vydac 218TP510 semipreparative column (The Separation Group, Hesperia, CA) previously equilibrated in solvent A (10 mM trifluoroacetic acid, TFA; Fluka), at a flow rate of 5 ml/min as described elsewhere (Abriouel et al., 2003). The concentration of purified derivatives, determined by measuring UV absorption at 280 nm in a Nanodrop, was converted to protein concentration using molecular extinction coefficients calculated from the contributions of individual amino acid residues introduced (Gill and von Hippel, 1989). "
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    ABSTRACT: The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer. Copyright © 2015 Elsevier Inc. All rights reserved.
    Journal of Structural Biology 03/2015; 190(2). DOI:10.1016/j.jsb.2015.03.006 · 3.23 Impact Factor
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    • "Theoretical isoelectric point (pI), molecular weight, amino acid composition, grand average hydropathicity (GRAVY), estimated half-life, extinction coefficient (Gill and Von, 1989), instability index (Guruprasad et al., 1990), aliphatic index (Ikai, 1980) of the protein were calculated using the preset parameters through protparam. Properties like solvent accessibility, transmembrane helices, globular regions, bend region, random coil and coiled-coil region were predicted by SOPMA. "
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    Molecular Immunology 02/2015; 65(1):189–204. DOI:10.1016/j.molimm.2014.12.013 · 2.97 Impact Factor
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