[Fig.3(B)].3(B)]. The degree of cleavage was measured by estimating the intensity of the band (on poly-acrylamide gel) corresponding to aFGF at 16 kDa (remaining after trypsin digestion) using a scanning densitometer. However, the maximum percentage of protein precipitated, in the TCA concentration range of 1545% w/v, is only about 70% [Fig. S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor. Dosztanyi Z, Tompa P. Prediction of protein disorder. The trichloro moiety is important for its protein precipitation capability of TCA. Effect of trichloroacetic acid. The new PMC design is here! 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. It is important to understand if proteins precipitated by TCA can be completely recovered in solution in their native conformation. Existence of molten globule-like state. 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. Unless specified, all solutions were prepared in 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. The crosspeaks that show significant decrease in their intensities represent the STCA binding sites. The protease action was stopped by heating the mixture (protein + trypsin) at 90C for 10 min. The flow rate of elution was 1 mL/min. All other chemicals used were of high-quality analytical grade. aFGF, in its native conformation (at pH 7.0), elutes as a single peak with an elution time of 83 1.0 min [Fig. 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. The HSQC spectra of aFGF acquired beyond 10% w/v STCA show limited chemical shift dispersion and are typical of denatured states of proteins (date not shown). [Fig.8(D)].8(D)]. [Fig.6(B-c)].6(B-c)]. Hongbo X, Slobodan V, Lilia MI, Oldfield CJ, Dunker KA, Uversky VN, Obradovic A. Functional anthology of intrinsic disorder. Panel (A) SDS-PAGE analysis of the limited trypsin digestion products of aFGF in the native and in the MG- like state(s) in 5% w/v STCA. FOIA Cortese MS, Uversky VN, Dunker KA. 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. The 308/350 nm emission ratio for aFGF in its native conformation is 4.8 [Fig. Ahmad A, Madhusudanan KP, Bhakuni V. Trichloroacetic acid and trifluoroacetic acid-induced unfolding of cytochrome c: stabilization of a native-like folded intermediate. Residues that show significant decrease in intensity and chemical shift perturbation in presence of 5% w/v STCA are depicted in green. Lane M shows the molecular weight marker. [Fig.5(A)].5(A)]. Radivojac P, Iakoucheva LM, Oldfield CJ, Obradovic A, Uversky VN, Dunker AK. All fluorescence spectra were collected on a Hitachi F-2500 spectrofluorometer at 2.5 nm resolution, using an excitation wavelength of 280 nm. The results discussed so far clearly suggest that TCA-induced protein precipitation is a reversible association reaction. Sagar AJ, Pandit MW. Heparin-sepharose was obtained from Amersham Pharmacia Biotech. Recombinant aFGF was prepared from transformed Escherichia coli BL21(DE3)pLysS. Labeled 15NH4Cl and D2O were purchased from Cambridge Isotope Laboratories. Hydrochloroacetic acid did not precipitate aFGF in the concentration range 090% v/v [Fig. I. 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. Kathir KM, Ibrahim K, Rajalingam D, Prudovsky I, Yu C, Kumar TKS. de Roos B, Duthie SJ, Polley AC, Mulholland F, Bouwman FG, Heim C, Rucklidge GJ, Johnson IT, Mariman EC, Dnaiel H, Elliott RM. In our opinion, the findings of this study are expected to pave way for the development of more efficient protein extraction and sample preparation protocols, which are a prerequisite for reliable proteomic analysis. 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. Urea-induced equilibrium unfolding of aFGF, monitored by changes in the intrinsic tryptophan florescence, shows that the majority of the protein exists in the native conformation at denaturant (urea) concentrations lower than 1M [Fig. The 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. Protein concentrations in the supernatant were determined using the respective molar extinction coefficients of the proteins at 280 nm. [Fig.4(B)].4(B)]. Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates. and transmitted securely. Panel (A) Fluorescence spectrum of aFGF, (a) extracted in 10 mM tris (pH 7.5), and (b) extracted in 10 mM tris (pH 7.5) containing 500 mM sodium bicarbonate. Intrinsic disorder in scaffold proteins: getting more from less. Tricholoacetic acid is the most acidic of the all the choloroacetic acids used. Isaacson T, Damasceno CM, Saravanan RS, He Y, Catala C, Saladie M, Rose JK. The concentration of the protein used in the experiment was 0.5 mg/mL. 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. [Fig.8(A)].8(A)]. Uniform 15N labeling aFGF was achieved using M9 minimal medium containing 15NH4Cl. Two closely eluting peaks at 61 min and 64 min were observed in 5% w/v STCA. Undigested aFGF yields a band on SDS-PAGE, which corresponds to a molecular mass of about 16 kDa [Fig. In this context, the protein precipitating action of acids stronger than TCA such as, trifluoroacetic acid, tribromoacetic acid, perchloric acid, and hydrochloric acid were examined. [Fig.8(C)].8(C)]. Bicarbonate ion possibly appears to reverse the precipitation action of TCA by neutralizing the residual acidic trichoroacetate ions remaining (bound to the protein precipitate) even after extensive washing with acetone. sharing sensitive information, make sure youre on a federal 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. The spectra were processed on a Windows workstation using Xwin-NMR and Sparky softwares.39, National Library of Medicine [Fig.8(B)].8(B)]. The acid-induced denaturation experiments were monitored by far-UV CD (at 228 nm) spectroscopy (AVIV-215 spectropolarimeter) at 25C. Kumar TKS, Subbiah V, Ramakrishina T, Pandit MW. In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome. Panel (B) Percentage of the 16 kDa aFGF band remaining after incubation with trypsin for various intervals of time. [Fig.2(C)].2(C)]. 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. 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. In this context, it would be of interest to note that Cortese et al.,31 observed that unstructured proteins in E. coli have a lower tendency to precipitate in TCA. Panel (B) Plot of ellipticity (at 228 nm) versus concentration of STCA. The precipitated products of the reaction were analyzed by SDS-PAGE. S2). The unfolding profile was monitored by the changes in the emission intensity at 350 nm. will also be available for a limited time. [Fig.6(A)].6(A)]. 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. The proteins, aFGF, and lysozyme were treated with TCA and other acids using the method of Sagar et al.38 Protein solutions containing appropriate concentrations of the respective acids were prepared by the addition of requisite amounts of the stock solutions of the individual acids to aqueous solutions of proteins. [Fig.2(A)].2(A)]. Weist S, Eravci M, Broedel O, Fuxius S, Eravci S, Baumgartner A. The .gov means its official. The partially structured state(s) that accumulates in low trichloroacetate concentrations (5% w/v STCA/TCA) closely resembles the acid intermediate (A-state) identified in several proteins.36 The A-states are generally very sticky and show a high-tendency to irreversibly aggregate.36,37 However, in marked contrast, the trichloroacetate-induced precipitate is a reversible association of proteins in their partially structured intermediate state(s). [Fig.7(C)].7(C)]. Da Cruz S, Martinou JC. The percentage of undigested aFGF was estimated from the intensity of the 16 kDa Coomassie blue stained band in the polyacrylamide gel. An official website of the United States government. Hsieh SY, Zhuang FH, Wu YT, Chen JK, Lee YL. Accessibility about navigating our updated article layout. Therefore, it can be argued that the protein precipitation is not specific to TCA, but in principle can be achieved using any strong acid. [Fig.6(B-a)].6(B-a)]. The flow rate of elution was 1 mL/min at 25C. Changes in the emission intensity at 520 nm and the shift in wavelength emission maximum on ANS are represented in closed and open circles, respectively. 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. Therefore, subtle conformational changes that occur in a protein during equilibrium unfolding/refolding can be easily monitored by the limited proteolytic digestion technique. It takes more than 3 h before the protein solution turns turbid. The slow precipitation of proteins and the near-neutral nature of STCA provides an avenue to probe the structural transitions that precede the precipitation of proteins induced by TCA. Schulman BA, Kim PS, Dobson CM, Redfield C. A residue-specific NMR view of the non-cooperative unfolding of a molten globule. However, the maximum amount of protein precipitated in TFA was significantly lower than that observed using TCA [Fig. The Cm value for the urea-induced unfolding of the aFGF, estimated from the fluorescence experiments, is 2.1 0.05M. Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis. Panel (A) shows the far- UV CD spectra of aFGF at different concentrations of STCA. Protein peaks were detected by their 280 nm absorbance. [Fig.4(A)].4(A)]. 8600 Rockville Pike 15N decoupling during data acquisition was accomplished using the globally optimized altering phase rectangular pulse sequence, and 2048 complex data points were collected in the 15N dimension. 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. Blank corrections were made in all the spectra using 10 mM phosphate buffer containing 100 mM NaCl and respective concentrations of STCA. Urea-induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. The 1H-15N HSQC spectrum is a fingerprint of the backbone conformation of a protein under given experimental conditions.351H-15N HSQC of aFGF in 0% (w/v) STCA is well-dispersed indicating that the protein is well-structured in its native conformation [Fig. However, in the partially unfolded state(s) induced in 5% w/v STCA, band representing the intact aFGF molecule completely disappears within 5 min of incubation of the protein with trypsin [Fig. [Fig.5(A)].5(A)]. Before 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. 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. The protein in 5% STCA unfolds completely in urea concentrations greater than 1.8M [Fig. [Fig.1AC)].1AC)]. The purity of the protein was assessed using SDS-PAGE. Panel (A) Binding of ANS to aFGF at various concentrations of STCA. Elution of the protein was monitored by its absorbance at 280 nm. 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 dye exhibits weak binding to both the native and extensively unfolded states of proteins.19,20 However, it binds quite strongly to solvent-accessible hydrophobic pockets in stable intermediates such as the molten globule state(s).20 In this context, the binding affinity of aFGF to ANS was monitored at different concentrations of STCA (090% w/v) [Fig. [Fig.2(B)].2(B)]. [Fig.3(B)].3(B)]. Urea-induced equilibrium unfolding of aFGF in 5% w/v STCA was monitored by steady-state fluorescence. The Lane-1 represents the trypsin digestion products after various time periods of incubation of native aFGF with trypsin. 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. These results suggest that the trichloroacetate moiety is important for protein precipitation. Protein precipitation profiles obtained in different acids revealed that acetic acid and chloroacetic acid did not significantly precipitate aFGF. Sivaraman T, Kumar TKS, Jayaraman G, Yu C. The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation. Final spectra were obtained after correcting for the background fluorescence of ANS. S1), suggesting that the TCA is less efficient in precipitation proteins in their denatured states. Intrinsic disorder and functional proteomics. 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. At low concentrations, the negatively charged tricholoroacetate ions plausibly trigger protein unfolding by disrupting the electrostatic interactions that stabilize the native conformation of proteins. government site. Necessary background corrections were made in all spectra. In summary, these results clearly suggest that the TCA-induced precipitation profiles are independent of the native conformation of proteins. Cortese MS, Baird JP, Uversky VN, Dunker AK. 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. Development and application of a two-phase, on-membrane digestion method in the analysis of membrane proteome. 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. Panel (A) Overlap of 1H-15N HSQC spectrum of aFGF in absence (red) and presence of 5% w/v STCA (green). These results clearly suggest that STCA (like TCA) induced protein precipitation involves the reversible association of protein molecules. The partially structured intermediate accumulated in 5% w/v STCA/TCA is largely responsible for the protein precipitation reaction. Cross-peaks corresponding to Cys30, His35, Ile56, Leu86, Asn114, Lys115, Trp121, Phe122, Lys126, Lys132, Gly134, and Tyr139 completely disappeared in the 1H-15N HSQC spectrum of aFGF obtained in 5% (w/v) STCA [Fig. Xu A, Xie Q, Zhou HM. In general, the decrease in cross-peak intensity of selective residues is ascribed to faster internal mobilities of residues in the flexible regions of the protein molecule. Such precipitation-competent intermediate(s) plausibly do not accumulate in the unfolding pathway(s) of some of other acids, and therefore these acids are unable to mimic the protein-precipitation action of TCA. [Fig.2(C)].2(C)]. Of these acids, only TFA showed a strong tendency to cleave the protein [Fig. The extent of 15N labeling was verified by ES-Mass analysis. No precipitate was observed immediately after addition of STCA in the concentration range of 090% w/v. 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). [Fig.55(B)]. Zhou J, Lin Y, Deng X, Han H, Shi W, Li Y. The amount of protein precipitate formed in different concentrations of TCA (090% w/v) was monitored using SDS-PAGE and by measuring protein absorbance at 280 nm [Fig. 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. The acid(s) treated proteins solutions were incubated at 25C for 1 h. The precipitated proteins samples were pelleted down by centrifugation at 12,000 rpm for 20 min. The results of the ANS binding and the SEC experiments analyzed in conjunction, clearly suggest that a MG-like intermediate state(s) maximally accumulates in 5% (w/v) STCA. The authenticity of the truncated sample was verified by ES-Mass analysis. Biological processes and functions of proteins with long disordered regions. The TCA precipitation profile of aFGF, monitored using SDS PAGE, is also bell-shaped [Fig. (a) 0% w/v STCA, (b) 5% w/v STCA, and (c) 50% w/v STCA. Subsequently, several other studies suggested that the acidic nature of TCA is important for the conformational changes that trigger protein precipitation.1921 However, the mechanism by which TCA precipitates proteins is not clearly understood. The broad minor peak (with an average retention time of 40 min) observed in the FPLC profile possibly represents the population of oligomeric species formed in the TCA-induced protein precipitate. The 308/350 nm emission ratio reached a maximum value (3.6) when the extraction was performed in 10 mM tris containing 500 mM sodium bicarbonate [Fig. The protein in the absence of STCA shows weak emission at 520 nm. PMC legacy view Similar trends were observed using lysozyme (Supporting Fig. [Fig.8(A)].8(A)]. In addition, TCA appears to be less effective in precipitating proteins in the disordered/unfolded state(s). In the 1960s, Charles Tanford18 proposed that TCA forces protein to precipitate by sequestering the protein-bound water. TCA is significantly less effective in precipitating unfolded states of proteins. Blank corrections were made in all of the spectra using 10 mM phosphate buffer containing 100 mM NaCl. Protein precipitation curves obtained by monitoring the intensity of the Coomassie-stained protein bands on SDS gels are bell-shaped [Fig. Trifluoroacetic acid (TFA), trobromoacetic acid (TBA), and perchloric acid (PA) precipitated aFGF. STCA-induced secondary structural changes in aFGF monitored by far-UV CD. Size exclusion chromatogram of the protein sample extracted in 500 mM bicarbonate showed only a major peak (retention time 81) corresponding to the native conformation of aFGF [Fig. Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor. [Fig.6(B-c)].6(B-c)]. Ingredients for Luria Broth were obtained from AMRESCO. [Fig.2(A),2(A), inset]. To realize maximal expression yields, the composition of the M9 medium was modified by the addition of a mixture of vitamins. [Fig.6(A)].6(A)]. Learn more Panel (C) The percentage of protein precipitated in the presence of different concentrations of urea, 0M urea- closed circle, 6M urea- open circle, was measured based on the intensity (after Coomassie blue staining) of the 16 kDa protein band (on the polyacrylamide gel). Careers, Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, Charles Loftis's current address is: Langston University, OK 73050. Recent studies show that a significant percentage of proteins in the eukaryotic proteome are intrinsically disordered.22,23 It is believed that presence of high proportion of unstructured/disordered proteins in eukaryotes is related to the increased prevalence of signaling and regulation coupled with the fact that signaling functions are enriched in disorder.24 Typically, intrinsically disordered proteins exhibit high-solubility even under extreme conditions due to the presence of large proportion of charged residues and extended backbone structure.25,26 In this background, it will be interesting to examine the effectiveness of TCA to precipitate proteins in their completely denatured state(s).
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