Article

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
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Peter H. von Hippel, Aug 20, 2015
5 Followers
 · 
1,076 Views
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Recent concerning facts of Chikungunya virus (CHIKV); a Togaviridae family alphavirus has proved this as a worldwide emerging threat which causes Chikungunya fever and devitalizing arthritis. Despite severe outbreaks and lack of antiviral drug, a mere progress has been made regarding to an epitope-based vaccine designed for CHIKV. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites by using various immunoinformatics and docking simulation tools. Initially, whole proteome of CHIKV was retrieved from database and perused to identify the most immunogenic protein. Structural properties of the selected protein were analyzed. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell were checked for the selected protein. The peptide region spanning 9 amino acids from 397 to 405 and the sequence YYYELYPTM were found as the most potential B cell and T cell epitopes respectively. This peptide could interact with as many as 19 HLAs and showed high population coverage ranging from 69.50% to 84.94%. By using in silico docking techniques the epitope was further assessed for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post therapeutic strategy, three dimensional structure was predicted along with validation and verification that resulted in molecular docking study to identify the potential drug binding sites and suitable therapeutic inhibitor against targeted protein. Finally, pharmacophore study was also performed in quest of seeing potent drug activity. However, this computational epitope-based peptide vaccine designing and target site prediction against CHIKV opens up a new horizon which may be the prospective way in Chikungunya virus research; the results require validation by in vitro and in vivo experiments. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Molecular Immunology 02/2015; 65(1):189–204. DOI:10.1016/j.molimm.2014.12.013 · 3.00 Impact Factor
  • Source
    • "The stability of PrP C under these conditions was ensured by the absence of aggregate formation and isoform conversion, tested by light scattering and circular dichroism measurements. Magnetic separation was then performed and the supernatant was used to estimate the amount of immobilized PrP, based on the molar extinction coefficient of PrP (63,495 cm −1 M −1 at 280 nM [34]). The PrP-coated MBs were incubated with 1 M glycine or 1 M hydroxylamine solution for 30 min at 4 • C to quench all residual aldehyde groups. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Prion diseases are characterized by protein aggregation and neurodegeneration. Conversion of the native prion protein (PrPC) into the abnormal scrapie PrP isoform (PrPSc), which undergoes aggregation and can eventually form amyloid fibrils, is a critical step leading to the characteristic pathomorphologial hallmark of these diseases. However, the mechanism of conversion remains unclear. It is known that ligands can act as cofactors or inhibitors in the conversion mechanism of PrPC into PrPSc. Within this context, herein, we describe the immobilization of PrPC onto the surface of magnetic beads and the morphological characterization of PrPC-coated beads by fluorescence confocal microscopy. PrPC-coated magnetic beads were used to identify ligands from a mixture of compounds, which were monitored by UHPLC-ESI-MS/MS. This affinity-based method allowed the isolation of the anti-prion compound quinacrine, an inhibitor of PrP aggregation. The results indicate that this approach can be applied to not only “fish” for anti-prion compounds from complex matrixes, but also to screening for and identify possible cellular cofactors involved in the deflagration of prion diseases.
    Journal of Chromatography A 12/2014; http://dx.doi.org/10.1016/j.chroma.2014.12.014. DOI:10.1016/j.chroma.2014.12.014 · 4.26 Impact Factor
Show more