[Fig.4(A)].4(A)].
Panel (B) Plot of ellipticity (at 228 nm) versus concentration of STCA. Urea-induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. Biological processes and functions of proteins with long disordered regions. Buchanan S. Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. The potency of an acid to precipitate proteins appears to depend not only on the nature of the anion but also on its ability to stabilize the precipitation-competent intermediate(s). A protein extraction method compatible with proteomic analysis for the euhalophyte. The 308/350 nm emission ratio for aFGF in its native conformation is 4.8 [Fig. Size exclusion chromatography profile of the 5% w/v STCA-induced precipitate of aFGF, extracted in 10 mM tris (pH 7.2), showed three prominent peaks corresponding to molecular masses expected for the monomer, dimer, and trimer of the protein (Supporting Fig. Urea-induced equilibrium unfolding, under various conditions, was monitored by intrinsic tryptophan fluorescence. These results show that STCA is a protein denaturant, and the unfolding of aFGF is complete in concentrations of STCA greater than 20% w/v [Fig. sharing sensitive information, make sure youre on a federal ; and (4) What is the general mechanism underlying TCA-induced protein precipitation? The protease action was stopped by heating the mixture (protein + trypsin) at 90C for 10 min. Size-exclusion chromatography profile of aFGF extracted in 10 mM tris (pH 7.2), showed a peak (retention time 82 min) corresponding to the native conformation, and several other peaks with shorter retention times [Fig. The results discussed so far clearly suggest that TCA-induced protein precipitation is a reversible association reaction.
Similar trends were observed using lysozyme (Supporting Fig. Peterson GL. aFGF is a 16 kDa -barrel protein, containing a single tryptophan residue at position 121 of its amino acid sequence.27,28 Interestingly, despite the presence of the lone tryptophan, the fluorescence spectrum of aFGF, in its native state, is dominated by tyrosine emission at 308 nm [Fig. These results clearly suggest that unfolded/denatured proteins have a lower tendency to precipitate in TCA. In the absence of STCA, 40% of the band corresponding to undigested aFGF remains after 10 min of incubation with trypsin [Fig. Received 2008 Sep 22; Revised 2009 Feb 23; Accepted 2009 Feb 25. protein precipitation, NMR spectroscopy, molten globule-like state(s), protein isolation, proteomics, fibroblast growth factors. The protein precipitation effects of TCA were studied on four proteins [lysozyme, acidic fibroblast growth factor (aFGF), carbonic anhydrase, and bovine serum albumin] with varying isoelectric points and molecular masses. An official website of the United States government. The intensity of the bands corresponding to the untreated aFGF was considered as a control for 100% protection against trypsin action. Lane: M, Marker; 1, 0% w/v TCA; 2, 5% w/v TCA; 3, 10% w/v TCA; 4, 15% w/v TCA; 5, 20% w/v TCA; 6, 25% w/v TCA; 7, 30% w/v TCA; 8, 35% w/v TCA; 9, 40% w/v TCA; 10, 45% w/v TCA; 11, 50% w/v TCA; 12, 55% w/v TCA; 13, 60% w/v TCA; 14, 65% w/v TCA; 15, 70% w/v TCA; 16, 75% w/v TCA; 17, 80% w/v TCA; 18, 85% w/v TCA; and 19, 90% w/v TCA, respectively. Panel (B) The percentage of protein precipitated in TCA (open circle), dichloroacetic acid (filled circle), perchloric acid (open square), hydrochloric acid (filled square), tribromoacetic acid (filled triangle), trifluoroacetic acid (open triangle), and sodium trichloroacetate (X), was measured based on the intensity (of the Coomassie blue stained) of the 16 kDa protein band (on the polyacrylamide gel). Kathir KM, Ibrahim K, Rajalingam D, Prudovsky I, Yu C, Kumar TKS.
Hsieh SY, Zhuang FH, Wu YT, Chen JK, Lee YL. The precipitated products of the reaction were analyzed by SDS-PAGE. It takes more than 3 h before the protein solution turns turbid. The unfolding profile was monitored by the changes in the emission intensity at 350 nm.
All fluorescence spectra were collected on a Hitachi F-2500 spectrofluorometer at 2.5 nm resolution, using an excitation wavelength of 280 nm. The flat portion of the curve (Phase 2), observed in the TCA concentration range of 545% w/v, represents the maximum amount of protein precipitated [Fig. Protein concentrations in the supernatant were determined using the respective molar extinction coefficients of the proteins at 280 nm. Sivaraman T, Kumar TKS, Jayaraman G, Yu C. The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation. In the first phase (Phase 1), which occurs below 5% w/v TCA, increase in the acid concentration results in a progressive increase in the amount of protein precipitated [Fig. S2).
The inset figure shows the emission spectra of the aFGF at different concentrations of STCA. PMC legacy view These results suggest that the trichloroacetate moiety is important for protein precipitation. Panel (B) 1H-15N chemical shift perturbation of residues in aFGF in the presence of 5% w/v STCA. The spectra were processed on a Windows workstation using Xwin-NMR and Sparky softwares.39, National Library of Medicine In this context, in this study, we make an attempt to provide a comprehensive understanding of the mechanism underlying the TCA-induced protein precipitation. [Fig.6(A)].6(A)]. In our opinion, the mechanism proposed in this study will provide valuable clues for the development of efficient protocols to capture the entire proteome. Of these acids, only TFA showed a strong tendency to cleave the protein [Fig. Panel (C) Urea-induced equilibrium unfolding profile of aFGF, native state (filled circle) and in MG- like state in 5% w/v of STCA (open circle). Many cross-peaks show significant chemical shift perturbation and some disappeared in the 1H-15N HSQC spectrum of the aFGF acquired in 5% w/v STCA [Fig.
[Fig.1(B)].1(B)]. Wang X, Li X, Deng X, Han H, Shi W, Li Y.
All protein samples were prepared in 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. [Fig.2(C)].2(C)]. Panel (B)The percentage of TCA-induced protein precipitation of lysozyme (open circle), aFGF (filled square), carbonic anhydrase (open square), and BSA (open triangle) was measured based on the intensity (after Coomassie blue staining) of the bands of the corresponding proteins on the polyacrylamide gel. Recombinant aFGF was prepared from transformed Escherichia coli BL21(DE3)pLysS. Da Cruz S, Martinou JC.
Peaks representing the native (elution time, 85 0.5 min) and denatured states (elution time, 48 0.5 min) of aFGF were not observed in the elution profile obtained at 5% (w/v) STCA [Fig. Cross-peaks of some other residues corresponding to Leu28, Tyr29, Val45, Ser72, Meth81, Leu98, Lys115, Trp121, Lys126, Lys132, Gln141, Ala143, Ile144, Leu145, Phe146, Lys148, and Leu149 show significant 1H-15N chemical shift perturbation [Fig. It is not unreasonable to conceive that the observed longer elution times of the aFGF fractions are due to the highly compact nature of the partially unfolded stable intermediate states that accumulate in 5% w/v STCA. Weist S, Eravci M, Broedel O, Fuxius S, Eravci S, Baumgartner A.
ANS is a hydrophobic dye that is routinely used to detect the accumulation of stable intermediates in the unfolding/refolding pathways of proteins. The ellipticity band intensity (at 228 nm) progressively decreases with the increase in STCA concentration, suggesting progressive unfolding of the protein. At low concentrations, the negatively charged tricholoroacetate ions plausibly trigger protein unfolding by disrupting the electrostatic interactions that stabilize the native conformation of proteins.
Far UV circular dichroism (CD) spectrum of aFGF in its native conformation shows a strong positive ellipticity band (between 225 and 230 nm) typical of -barrel proteins [Fig. I. Hydrochloroacetic acid did not precipitate aFGF in the concentration range 090% v/v [Fig. [Fig.4(A)].4(A)]. The Lane-1 represents the trypsin digestion products after various time periods of incubation of native aFGF with trypsin. The TCA precipitation profile of aFGF, monitored using SDS PAGE, is also bell-shaped [Fig. Sample extraction techniques for enhanced proteomic analysis of plant tissues. These results clearly suggest that the acid nature of TCA does not solely contribute to its protein precipitation property [Fig. Two closely eluting peaks at 61 min and 64 min were observed in 5% w/v STCA. [Fig.8(D)].8(D)]. The absence of the preunfolding baseline (in the unfolding curve obtained using relative fluorescence intensity at 350 nm) precludes the accurate estimation of the parameters of unfolding. Li X, Xu S, Pan C, Zhou H, Jiang X, Zhang Y, Ye M, Zou H. Enrichment of peptides from plasma for peptidome analysis using multiwalled carbon nanotubes. aFGF, in its native conformation (at pH 7.0), elutes as a single peak with an elution time of 83 1.0 min [Fig. This study represents an attempt to provide a comprehensive understanding of the general mechanism(s) underlying the TCA-induced protein precipitation. We studied the protein precipitation action of sodium trichloroacetate under neutral conditions (dissolved in 100 mM phosphate buffer, pH 7.2) to understand the relative contribution(s) of the trichloroacetate moiety in protein precipitation. Cortese MS, Baird JP, Uversky VN, Dunker AK. The .gov means its official. It is important to mention that such partially unfolded intermediate(s) also accumulate in the STCA-induced unfolding pathway of other proteins, such as lysozyme (Supporting Fig. STCA-induced secondary structural changes in aFGF monitored by far-UV CD. Dosztanyi Z, Tompa P. Prediction of protein disorder. In summary, these results clearly suggest that the TCA-induced precipitation profiles are independent of the native conformation of proteins. Therefore, it can be argued that the protein precipitation is not specific to TCA, but in principle can be achieved using any strong acid. Increase in the TCA concentration(s), beyond 45% w/v (Phase 3), results in a sharp decrease in the amount of protein precipitated, and practically very little or no precipitate is observed beyond 60% w/v TCA [Fig. Partial unfolding of proteins results in the exposure of solvent-accessible nonpolar surface(s), and which consequently results in intermolecular coalescence of protein molecules leading to their precipitation. [Fig.1(C)].1(C)]. All the UV spectrophotometric measurements were carried out on a Hitachi-3300 spectrophotometer. Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment. The secondary structural elements in aFGF include 12 beta strands arranged into a -trefoil motif.
Proteins in the MG state(s) generally have persistent native secondary structural interactions and higher flexibility of the side chains due to loss in some tertiary structural interactions.33 Limited proteolytic digestion is an immensely useful technique to probe gross conformational flexibility of proteins in various conformational states.34 In general, proteolytic digestion is not only dependent on the stereochemistry and accessibility of the protein substrate but also on the specificity of the proteolytic enzyme. The partially structured intermediate accumulated in 5% w/v STCA/TCA is largely responsible for the protein precipitation reaction. The concentration of the protein was estimated based on the extinction coefficient value of the protein at 280 nm. [Fig.7(B)].7(B)]. Further increase in the STCA concentration not only results in a progressive decrease in ANS intensity at 520 nm but also is accompanied by a continuous red shift in the wavelength of maximum emission of the dye. Unlike TCA, precipitation of proteins in STCA is not instantaneous. The amino acid residues in aFGF that showed significant chemical shift perturbation and involved in STCA binding are indicated by their single letter codes. Extrapolation of the observed retention times to the standard plot (obtained using proteins of known molecular weights), reveals that peaks with retention times of 37 min and 42 min possibly correspond to dimeric and trimeric forms of aFGF, respectively. TCA-induced protein precipitation curves are U-shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. The nativity of aFGF recovered in solution from the TCA precipitate was measured using the ratio of emission at 308 and 350 nm. [Fig.1(B)].1(B)]. [Fig.8(A)].8(A)]. Carpentier SC, Witter E, Laukens K, Deckers P, Swennen R, Panis B. 8600 Rockville Pike [Fig.3(A)].3(A)]. In this study, we address these questions using aFGF as a model protein, and attempt to provide a comprehensive understanding of the molecular mechanism underlying the action of TCA on proteins. The concentration of the protein sample used was 0.1 mM in 90% H2O and 10% D2O prepared in 10 mM phosphate buffer containing 100 mM NaCl. To realize maximal expression yields, the composition of the M9 medium was modified by the addition of a mixture of vitamins. Protein peaks were detected by their 280 nm absorbance. S2). For the unfolding experiments, protein samples were dissolved in appropriate concentrations of urea prepared in 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. It should be important to mention that the percentage of protein(s) precipitated in various concentrations of TCA is independent of the protein concentration (in the range of 5 to 250 M) used. Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives. [Fig.4(B)].4(B)]. ; (2) Can TCA precipitate proteins in their unfolded/disordered conformation(s)? Nandakumar MP, Cheung A, Marten MR. Proteomic analysis of extracellular proteins from, Jacobs DI, van Rijssen MS, van der Heijden R, Verpoorte R. Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured. Digestion experiments were carried out by independently incubating aFGF in the presence of trypsin in 5% (w/v) STCA (at 1:1 protein to trypsin molar ratio) dissolved in 10 mM phosphate buffer containing 100 mM NaCl. Accessibility [Fig.7(A,B)].7(A,B)]. In addition, thermally unfolded aFGF also showed a decreased tendency to precipitate in TCA suggesting that the lower amounts of protein precipitation observed in 6M urea is not due to the interference of the denaturant in the TCA-induced precipitation reaction (data not shown). The concentration of urea required for 50% of the aFGF molecules (in 5% w/v STCA) to exist in the unfolded state(s) (Cm) is 1.2M. However, aFGF in the denatured state(s), in 50% (w/v) STCA, elutes relatively earlier (elution time 48 min) than in the native conformation [Fig. Careers, Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, Charles Loftis's current address is: Langston University, OK 73050. The lower stability of the STCA-induced MG-like intermediate is consistent with the loss of native tertiary structural interactions in the partially structured state. [Fig.4(B)].4(B)]. In addition, the wavelength of maximum emission of the dye shifts by about 10 nm from 506 nm to 496 nm when the protein is treated with 5% w/v STCA. Ishida T, Kinoshita K. Prediction of disordered regions in proteins based on the meta approach. Filled and open circles represent the percentage of aFGF (16 kDa band) cleaved in the native (in 0% STCA) and MG-like (in 5% w/v STCA) states, respectively. The authenticity of the truncated sample was verified by ES-Mass analysis. [Fig.7(C)].7(C)]. The purity of the protein was assessed using SDS-PAGE. Lane: M, Marker; 1, 0% w/v; 2, 5% w/v; 3, 10% w/v; 4, 15% w/v; 5, 20% w/v; 6, 25% w/v; 7, 30% w/v; 8, 35% w/v; 9, 40% w/v; 10, 45% w/v; 11, 50% w/v; 12, 55% w/v; 13, 60% w/v; 14, 65% w/v; 15, 70% w/v; 16, 75% w/v; 17, 80% w/v; 18, 85% w/v; and 19, 90% w/v.